To enhance the production of all-female populations in aquaculture, it is essential to understand the reproductive differences between neomales and normal males, since this knowledge can significantly impact the efficiency and success of producing all-female offspring. The objective of this paper was to perform a comparative analysis of sperm quality and fertilization potential between neomales (XX genotype) and normal males (XY genotype) in the bonylip barb (Osteochilus vittatus). Fifteen neomales and fifteen normal males, each weighing 200–400g, were used as experimental subjects, with their gonad development monitored monthly. To evaluate fertilization ability, fifty females (weighing 200–550g) were spawned with both neomales and normal males. Sperm velocity and motility were analyzed using computer-assisted semen analysis (CASA) and included parameters, such as linearity (LIN), straightness (STR), wobble (WOB), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), and percentage of motile sperm (% MOT). While there were no significant differences in sperm motility between neomales and normal males, the velocity parameters indicated that neomale sperm had higher percentages of straight-line velocity, linearity, straightness, and wobble. Significant differences (P< 0.05) were found in the amplitude of lateral head displacement and beat cross frequency values between the two groups. Additionally, sperm volume, fertilization rate, and hatching rate were significantly higher (P< 0.05) in neomales, with values of 14.33±2.08, 72.77±4.13, and 63.33±11.55, respectively. The findings suggest that neomales (genotype XX) in Osteochilus vittatus exhibit superior sperm quality and fertilization ability compared to normal males (genotype XY).