This study hypothesized whether the gender group composition in traditional learning (TL) versus collaborative learning (CL) classrooms of undergraduate biology majors and nonmajors correlate with students' achievements. We measured the effect on gender and the gender-specific achievements of the TL versus CL approach in single-gender and mixed-gender classes. A significant gender gap was found in the achievements of both nonmajor and major students. Females achieved higher grades in TL and CL sections in single-gender classes; overall, academic achievements between females (F) and males (M) demonstrated a significant difference at P<0.05. The effect size value between TL versus CL indicated that males benefited more than females implementing CL mixed-gender (2F+2M) in nonmajors and majors. While females in single-gender CL and TL classes performed higher than males, females performed relatively low in mix-gender CL (2F+2M). These findings indicate that gender-specific and context-specific learning pedagogies are required since they impact students' achievement.