This study examined seasonal variation and the effect of the transition period on physical fitness parameters in female youth soccer players. Twenty-four players (13–17 years old) were assessed through field and laboratory testing. They completed laboratory testing three times: (1) at the beginning of the season (post-preseason), (2) at the end of the regular season (postseason), and (3) following the transition period (preseason). Field testing was conducted post-preseason and postseason. Results indicated that weight and body fat significantly increased from post-preseason to postseason and following the transition period. A paired samples T-test revealed that the players performed significantly better in the vertical jump and 30 m sprint test (all p < 0.01) postseason compared to post-preseason. Also, it was indicated that torque production for the right quadriceps, left quadriceps, and right hamstring (all p < 0.01) was significantly reduced after the transition period by 10%, 11.8%, and 10.5%, respectively. Cardiorespiratory measurements demonstrated that performance on an incremental cardiopulmonary treadmill test, maximal oxygen consumption, velocity at the first ventilatory threshold, velocity at the second ventilatory threshold, and velocity at maximal oxygen consumption (all p < 0.01) were significantly reduced postseason and following the transition period. Based on the results, coaches and trainers should primarily focus on maintaining the aerobic capacity of the players during the season, as it has been indicated to be reduced from the beginning to the end of the competitive period (VO2max reduced by 3.3%). In addition, they should focus on maintaining lower body strength and aerobic capacity during the transition period.