This study aimed to characterize muscle activity in male soccer players with a history of hamstring strain injuries (HSI) during accelerated sprinting. Thirteen patients each in the HSI group (history of HSI) and in the healthy group (with no history of HSI) were included. 26 male soccer players of which 13 with and 13 without HSI history were included in this study. Ten muscles were evaluated on electromyography activity during overground sprinting. The testing protocol consisted of a maximal sprint over a distance of 30 meters. One running stride was divided into the early stance phase, late stance phase, early swing phase, mid-swing phase, and late swing phase, and the average muscle activity per phase and the timing of the peak root-mean-square value appearance during each stride were calculated. Statistical analysis was performed using repeated-measures two-way ANOVA (group × phase), and multiple comparison tests were performed using the Bonferroni method when the interaction or main effect was significant. The statistical significance level was set at p < 0.05. Gluteus maximus (Gmax), gluteus medius (Gmed), and external oblique (EO) showed activity differences based on HSI history. Gmax was 30% lower, EO was 20% lower, and Gmed was 40% higher in HSI group. This study suggests that, despite previous findings that HSI is most likely during the late swing phase, the HSI group shows a higher injury risk in the early stance phase. This is due to differences in trunk and gluteal muscle activity between the late swing and early stance phases compared to the healthy group. In summary, HSI group had lower activity in the muscles contributing to trunk instability, especially EO and Gmax, before and after ground impact during accelerated sprinting, compared to Healthy.