Harkleroad turned pro on June 12, 2000, and was quickly billed as the next American tennis superstar. Her debut was at the ITF tournament in Largo, Florida, in 1999. The following year she played her first WTA qualifying event in Miami, Florida and her first Grand Slam at the U.S. Open. In 2001, she returned to the same events while improving her status on the ITF Circuit. In 2002 she won her first tour matches, reaching the second round at San Diego, Hawaii, and Bratislava, where she made her first doubles semi-final with partner María Emilia Salerni. She ended 2002 in the top 200 for the first time.
Her breakthrough year was in 2003, when at Charleston she defeated three top 20 players at the time (No. 16 Elena Bovina, No. 19 Meghann Shaughnessy, and No.9 Daniela Hantuchová), losing just 11 games along the way to reach her first tour semifinal before losing to Justine Henin-Hardenne. She became the lowest-ranked semifinalist (No. 101) in the event's history since unranked Jennifer Capriati reached the finals in 1990.
After that performance, on April 14, 2003 she broke out from No. 101 to climb to a No. 56 ranking. Subsequently she reached the semifinals again at Strasbourg and scored her second Top 10 win and second over Daniela Hantuchová at Roland Garros while reaching the 3rd round and for the second time winning past the first round at a grand slam. On June 9, 2003 she soared into the Top 50 at No. 39 and reached the final in doubles at the Japan Open in Tokyo.
In 2004, she made her career first Tour final (at Auckland) before losing to defending champion Eleni Daniilidou. For most of the 2005 season, Harkleroad sat out due to various injuries and an illness in the family, but the time she was on court was spent on the ITF Women's circuit winning 2 titles. She did however reach a tour doubles final at Quebec City.
Harkleroad was selected for the USA in the Hopman Cup after Venus Williams withdrew. She and partner Mardy Fish went 0–3 in the event. Harkleroad pushed Tatiana Golovin to 3 sets before losing 3–6,6–4,3–6. Harkleroad lost 3–6,0–6 to Nadia Petrova and 6–3,4–6,4–6 to Alicia Molik. Harkleroad started out the main WTA season in Hobart, Tasmania. She lost in the first round to Austrian Sybille Bammer 7–6(2),6–1. Bammer eventually defeated Serena Williams in the quarterfinals. She lost to Daniela Hantuchová in the third round of the Australian Open 7–6 (6),5–7,3–6. In the previous rounds, she beat Meng Yuan of China 6–3,6–0 and upset 17th seeded German Anna-Lena Grönefeld 6–2,6–2. Harkleroad struggled in the clay court season and finished at Roland Garros in the second round losing to Venus Williams 1–6,6–7(8–10). Harkleroad was down 1–6, 1–5 before rebounding and holding set points. During this match, Williams hit the fastest recorded women's main draw serve, a record which Venus broke again in the U.S. Open on August 28, 2007. In the grass court season, Harkleroad's worst, Harkleroad played the Liverpool International Tennis Tournament for the second straight year as her Wimbledon warm-up. In 2006, she lost in the final to Caroline Wozniacki. This year, she defeated Wozniacki in the final to win the event. Harkleroad lost to Roberta Vinci, a good grass-court player in the first round of Wimbledon. In the U.S. Open Series, Harkleroad failed to qualify at the Acura Classic but qualified in Los Angeles and reached the second round before falling to Roland Garros runner-up Ana Ivanović 4–6,2–6. At the U.S. Open (tennis), Harkleroad fell to Ioana Raluca Olaru 5–7,6–1,4–6. Harkleroad will be playing for the first time since the U.S. Open in San Francisco, where she won $50,000 at the ITF challenger for the second consecutive year. Harkleroad ended the year by winning a $75,000 challenger in Pittsburgh and a $50,000 challenger in La Quinta back to back. She also ended the year ranked 76, her second highest year end to date. Harkleroad also won the doubles title in la Quinta with Christina Fusano. Harkleroad finished with a 29–20 record in singles and a 13–11 record in doubles for the 2007 season.