Abstract: Objective Whitmania pigra is one of the original source species of medicinal leeches used in traditional Chinese medicine. It has become the dominant species in leech farming and stock enhancement in China, and enjoys promising market potential. To compare the differences in the production and reproductive traits, medicinal efficacy, and disease resistance among different populations of W. pigra. Methods This study conducted a comparative analysis of five wild geographical populations of W. pigra from Nanjing, Jiangsu (JS), Daxing, Beijing (BJ), Dezhou, Shandong (SD), Baodi, Tianjin (TJ), and Cangzhou, Hebei (HB), China. The evaluated parameters included the reproductive performance of parent W. pigra, average individual body mass, the specific growth rate, and survival rate of juvenile W. pigra, dry weight ratio of adult W. pigra, antithrombin activity of air-dried products adult W. pigra, and disease resistance after challenge with Citrobacter freundii. Results In terms of production traits, the JS population exhibited the highest body weight among wild parental W. pigra (13.86±4.51)g, which was extremely significantly higher than those of the TJ, HB, and SD populations (P<0.01), but showed no significant difference compared to the BJ population (12.50±3.30) g (P>0.05). The specific growth rates at 15 and 22 days of age in the JS population were higher than those in other populations. After 120 days of rearing, the JS population achieved the highest average individual body weight, whereas the TJ population had the lowest. The BJ population showed the highest survival rate, followed by the JS population, and the TJ population had the lowest. The processing yield of the JS population was the highest (16.39±2.48)%, significantly higher than those of the TJ and HB populations (P<0.05). Regarding reproductive performance, the BJ population had the highest cocoon hatching rate, while the JS population exhibited the highest average cocoon weight and average number of juveniles per cocoon; the TJ population consistently showed the lowest values for all three indices. In terms of medicinal efficacy, the air-dried adult W. pigra of the SD population demonstrated the highest thrombin inhibitory activity (8.33±0.30) U/g, whereas the HB population had the lowest (6.93±0.32) U/g. Concerning disease resistance, following artificial infection with Citrobacter freundii for 14 days, the JS population displayed the lowest cumulative mortality (60%) within 14 days, while the TJ population reached 100%. After infection, the total antioxidant capacity (T-AOC) level increased in all populations, whereas catalase (CAT) activity decreased to varying degrees compared with the control groups; acid phosphatase (ACP), alkaline phosphatase (ALP), and lysozyme (LZM) activities all increased. The activities of superoxide dismutase (SOD), CAT, and T-AOC level in the JS population were significantly higher than those in the TJ and HB populations (P<0.05), and the activities of ACP and LZM were significantly higher than those in the TJ population (P<0.05). Conclusion The JS population demonstrated selective breeding advantages in both production/reproductive traits and disease resistance, making it a suitable core foundational population for subsequent genetic improvement. The BJ and SD populations exhibited respective advantages in reproductive traits and medicinal quality, and could be utilized as auxiliary breeding populations. The TJ population performed poorly in production traits, reproductive performance, and disease resistance, indicating a need for enhanced germplasm improvement and genetic rejuvenation. This study provides a theoretical reference for the artificial propagation and selective breeding of W. pigra.