The number of chromosomes of Misgurnus anguillicaudatus (Cobitidae; Cobitinae; Misgurnus) is (2n=50) and that of Paramisgurnus dabryanus (Cobitidae; Cobitinae; Paramisgurnus) is (2n=48). Hybridization between M. anguillicaudatus (DD) and P. dabryanus (PP) can successfully obtain hybrid progeny(DP, PD) with karyotypes (2n=49), but the testes and ovaries of the hybrid progeny showed obvious developmental retardation. Self-crossing and backcrossing experiments of DP (DD♀×PP♂) showed that male DP were infertile. The sperm morphology and vitality of the loach M. anguillicaudatus (DD)， large scale loach P. dabryanus (PP), hybrid F₁ generation (DP) and (PD) were analyzed through computer assisted sperm analysis system (CASA), optical microscope, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and flow cytometry technology in order to explore the reason of male sterility in F₁ generation of hybrid between M. anguillicaudatus and P. dableurus. The results showed that the sperm motility rates of DD and PP were 76.50%±0.70% and 75.17%±8.60% respectively at 30 s of activation, which were significantly higher than DP (3.65%±1.75%) and PD (2.68%±0.63%) (P<0.01), and the related motion parameters such as VAP, VCL, VSL of hybrid loach are significantly lower than DD and PP (P<0.01), which reveals that the the sperm of the hybrid loach are particularly inactive. Ploidy identification was carried out on the sperms of four species of loach in the sperms of DD and PP by flow cytometers. It was found that the sperms of DD and PP were mostly 1N cells, while there were a large number of 2N and 4N and a small number of 8N cells in the sperms of DP and PD except for the normal haploid sperm. The spermatozoa in DD and PP testes were dense, intact and uniform in length (33.71±2.12) and (34.28±1.83) μm, respectively, by SEM and TEM. The head length of sperm in the testis of DP and PD was significantly longer than that of DD and PP (P<0.05), while the tail length, midlength and full length of sperm in the testis of DP and PD were significantly shorter than those of DD and PP (P<0.05). DP and PD in the testis displayed a large number of abnormal sperm, such as multitailed sperm, binuclear sperm and big-head sperm. Head size showed obvious polymorphism, according to the head size measurements to estimate its ploidy, including normal sperm haploid sperm, at the same time have diploid and tetraploid sperms, or even higher haploid sperm. The abnormal sperm of these hybrid loach provided important evidence for its low sperm motility and male sterility.