Samantha first played professional tennis in 1999 on the ITF circuit. In 2000 she debuted on the Women's Tennis Association tour where she lost in the first round of Australian Open qualifying to Louise Latimer. In 2001 she won four straight ITF titles (three in Japan and one in Australia). In 2002 she lost to Anastasia Myskina at the first round at the Gold Coast event; she was also given a Wildcard into the Australian Open where she lost in three sets to Greta Arn. For the rest of the 2002 season, Samantha played on the ITF Circuit.

In 2003 Samantha won her first WTA singles match on home soil where she reached the third round of the Australian Open, where she beat Conchita Martinez and Vanessa Webb in the first and second round respectivley. She lost in the third round to Daniela Hantuchová with a scoreline of 6-4 6-2. After her form in Australia, Samantha then went onto qualify for a WTA event in Memphis. She played more ITF events and failed to qualify for the Roland Garros losing in the first qualifying round to Wynne Prakusya. She was given a Wildcard into Wimbledon where she was unfortunate to meet Lindsay Davenport in the first round. Samantha put up a good fight but she lost 7-6 7-5.

In 2004 she reached the semi-final of the Gold Coast event where she made the semi-finals, beating Meghann Shaughnessy, Karolina Å prem and Magui Serna along the way, before falling to Ai Sugiyama, who would go on to win the tournament. The next week she would reach the second round of the WTA tournament in Hobart and then the second round of the Australian Open, losing to Aniko Kapros. She later successfully qualified for WTA events in Acapulco, Indian Wells, Vienna and Birmingham. She lost in the first round of Roland Garros and Wimbledon. She competed at the Athens Olympics where she lost in the first round of the tennis competetion to Chanda Rubin. She continued to play WTA qualifying events, successfully qualifying for the Japan Open and Bali in the autumn of 2004.

In 2005 she reached her first WTA tour final at the Gold Coast event, losing the final to Patty Schnyder; the next week she made her second final at Sydney losing to Alicia Molik. She was unlucky to meet Amelie Mauresmo in the first round of the Australian Open where she lost 6-2 6-3. However, she won the mixed doubles title with fellow Australian Scott Draper. In 2005 she won seven WTA doubles titles with her partners Bryanne Stewart (Sydney and Amelia Island) and Lisa Raymond (New Haven, US Open, Luxembourg, Moscow, Year Ending Championships).

Samantha finished the year ranked #46 in singles and #2 in doubles.

In 2006 she represented Australia along with Todd Reid at the Hopman Cup. She lost in the first round of Sydney to Nicole Vaidišová in three sets.

During the 2006 Australian Open, she made it to the fourth round, the highest round she has made it to ever in a Grand Slam event. She beat Sybille Bammer in the third round of Austria in three sets 7-5 4-6 6-3, then went on to lose to Martina Hingis 6-1 7-6 (8) in the round of sixteen. She previously had beaten Severine Beltrame and Ana Ivanovic in the first and second rounds.

In February, 2006, Samantha reached the quarter finals at the Tier I event in Tokyo, beating Aiko Nakamura and Daniela Hantuchova before falling to Maria Sharapova.

Seeded fifth in the WTA event in Memphis, USA, Samantha lost in the first round to American Ashley Harkleroad.

In Indian Wells in March, 2006, she reached the second round, beating Yulina Fedak in round one, before falling to ]'s Dinara Safina. In Miami, Samantha beat Dutch star Brenda Schaltz-McCarthy, before losing again to Amelie Mauresmo.

In April 2006, Stosur crashed out in round one of Charleston to rising Czech teenager Lucie Safarova.