Eccentric and concentric actions produce distinct mechanical stimuli and result in different adaptations in skeletal muscle architecture. Cycling predominantly involves concentric activity of the gastrocnemius muscles, while playing basketball requires both concentric and eccentric actions to support running, jumping, and landing. The aim of this study was to examine differences in the architecture of gastrocnemius medialis (GM) and gastrocnemius lateralis (GL) between elite basketballers and cyclists. A trained sonographer obtained three B-mode ultrasound images from GM and GL muscles in 44 athletes (25 basketballers and 19 cyclists; 24 ± 5 years of age). The images were digitized and average fascicle length (FL), pennation angle (θ), and muscle thickness were calculated from three images per muscle. The ratio of FL to tibial length (FL/TL) and muscle thickness to tibial length (MT/TL) was also calculated to account for the potential scaling effect of stature. In males, no significant differences were identified between the athletic groups in all parameters in the GM, but a significant difference existed in muscle thickness in the GL. In basketballers, GL was 2.5 mm thicker (95% CI: 0.7–4.3 mm, p = 0.011) on the left side and 2.6 mm thicker (95% CI: 0.6–5.7 mm, p = 0.012) on the right side; however, these differences were not significant when stature was accounted for (MT/TL). In females, significant differences existed in the GM for all parameters including FL/TL and MT/TL. Female cyclists had longer FL in both limbs (MD: 11.2 and 11.3 mm), narrower θ (MD: 2.1 and 1.8°), and thicker muscles (MD: 2.1 and 2.5 mm). For the GL, female cyclists had significantly longer FL (MD: 5.2 and 5.8 mm) and narrower θ (MD: 1.7 and 2.3°) in both limbs; no differences were observed in absolute muscle thickness or MT/TL ratio. Differences in gastrocnemius muscle architecture were observed between female cyclists and basketballers, but not between males. These findings suggest that participation in sport-specific training might influence gastrocnemius muscle architecture in elite female athletes; however, it remains unclear as to whether gastrocnemius architecture is systematically influenced by the different modes of muscle activation between these respective sports.