Background and Objective: Seropositivity rates of rubella virus infection among pregnant women vary worldwide. Yemen is among the developing countries the rubella disease is still present. This study was designed to estimate the seropositivity rate of rubella infection and risk factors associated among pregnant women attending antenatal care clinics in Mukalla city, Hadhramaut, Yemen. Materials and Methods: This was a cross-sectional study performed on a total of 190 pregnant women and the serum samples were collected and screened using the enzyme-linked immunosorbent assay (ELISA) anti-rubella virus IgM and IgG test. Qualitative demographic and reproductive data were collected using a standardized questionnaire. Results: One hundred-ninety pregnant women tested for rubella antibodies, 136 (71.6%), were positive for anti-rubella IgG, while 17 (8.9%) was positive for IgM. Higher seropositivity rates were found in the age group of the age groups 15-30 years with significantly dependent (COR = 0.749, 95% CI = 0.113-0.557, p = 0.001) and the moderate level income (COR = 0.761, 95% CI = 0.075-0.760, p = 0.015). Anti-IgM positive rubella infection had a significant relationship with the pregnant women miscarriage (COR = 0.925, 95% CI = 0.020-0.283, p = 0.00) and the risk of contracting rubella virus infection was found to increase with history of live births with a statistical significance (COR = 1.942, 95% CI = 1.020-3.695, p = 0.043). Conclusion: Seropositivity rates of a rubella infection are high in Mukalla city, Hadhramaut and are significantly associated with an increase in age and being income level. The risk of contracting rubella infection was found to increase with gestational age and associated with miscarriage. Screening of rubella and immunization of women at risk is highly recommended in this area with a high nonimmune rate against the rubella virus.