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Background: This study examined gastrocnemius medialis (GM) architectural properties and ankle joint range of motion (ROM) between female athletes with different flexibility training background. Methods: Elite rhythmic gymnasts (<i>n</i> = 10) were compared to national level volleyball athletes (<i>n</i> = 10). Fascicle length, pennation angle and muscle thickness at the medial and the distal part of GM, and ankle ROM were measured at rest and during 1 min of static stretching. Results: At rest, rhythmic gymnasts displayed longer fascicles compared to volleyball athletes, at the medial (5.93 &#177; 0.27 vs. 4.74 &#177; 0.33 mm, respectively, <i>p</i> = 0.001) and the distal part of GM (5.63 &#177; 0.52 vs. 4.57 &#177; 0.51 mm, respectively, <i>p</i> = 0.001), smaller pennation angle at the medial part (22.4 &#177; 2.5 vs. 25.8 &#177; 2.4&#176;; respectively, <i>p</i> = 0.001) and greater ankle angle (121.7 &#177; 4.1 vs. 113.2 &#177; 3.7&#176;, respectively, <i>p</i> = 0.001). During the 1 min of static stretching, gymnasts displayed greater fascicle elongation at the distal part (<i>p</i> = 0.026), greater maximal ankle dorsiflexion (<i>p</i> &lt; 0.001) and muscle tendon junction displacement (<i>p</i> &lt; 0.001) with no difference between groups in pennation angles (<i>p</i> &gt; 0.145), muscle thickness (<i>p</i> &gt; 0.105), and fascicle elongation at mid-belly (<i>p</i> = 0.063). Conclusions: Longer muscle fascicles at rest and greater fascicle elongation at the distal part of GM may contribute to the greater ankle ROM observed in rhythmic gymnasts.