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Sex differences in muscle fiber‐type composition have been documented in several muscle groups while the hamstring muscle fiber‐type composition has been poorly characterized. This study aimed to compare the semitendinosus muscle composition between men and women. Biopsy samples were obtained from the semitendinosus muscle of twelve men and twelve women during an anterior cruciate ligament reconstruction. SDH and ATPase activities as well as the size and the proportion of muscle fibers expressing myosin heavy chain (MyHC) isoforms were used to compare muscle composition between men and women. The proportion of SDH‐positive muscle fibers was significantly lower (37.4 ± 11.2% vs. 49.3 ± 10.6%, p < 0.05), and the percentage of fast muscle fibers (i.e., based on ATPase activity) was significantly higher (65.8 ± 10.1% vs. 54.8 ± 8.3%, p < 0.05) in men versus women. Likewise, men muscles exhibited a lower percentage of the area that was occupied by MyHC‐I labeling (35.6 ± 10.1% vs. 48.7 ± 8.9%; p < 0.05) and a higher percentage of the area that was occupied by MyHC‐IIA (38.3 ± 6.7% vs. 32.5 ± 6.5%; p < 0.05) and MyHC‐IIX labeling (26.1 ± 9.6% vs. 18.8 ± 8.5%; p = 0.06) as compared with women muscles. The cross‐sectional area of MyHC‐I, MyHC‐IIA, and MyHC‐IIX muscle fibers was 31%, 43%, and 50% larger in men as compared with women, respectively. We identified sex differences in semitendinosus muscle composition as illustrated by a faster phenotype and larger muscle size in men as compared with women. This sexual dimorphism might have functional consequences.