Blood group determination plays a critical role in medical science, genetics, and population studies. This study aimed to investigate the prevalence and gender association of blood groups in the Jalgaon district, located in the northern region of Maharashtra. A total of 2,010 individuals, comprising randomly selected males and females, participated in this research. A non-invasive methodology was employed, wherein data were collected solely through verbal communication. Subjects unaware of their blood group were excluded from the study to ensure data accuracy. The analysis revealed the distribution pattern of blood groups across the population, with statistical evaluation conducted using the chi-square test of independence to assess the association between gender and blood group prevalence. The findings provide insights into the gender-based prevalence of various blood groups within the district, contributing valuable information to regional genetics and medical research. Further implications of this study include its utility in healthcare planning, blood donation drives, and epidemiological studies.