Background information: HBV infection is a serious health problem and a leading cause for morbidity and mortality. Objective: To determine HBV markers ((Anti-HBcore (IgM), Anti-HBcore (IgG), HBsAb, HBsAg, and HBeAg)) among Village Midwifes in Khartoum State, Sudan, 2014. Method: It is a cross sectional study where 335 village midwives were surveyed. A pre-tested close ended questionnaire was used for demographic data. Five ml of venous blood was collected; sera were separated and stored at-20° centigrade for Eliza testing. All specimens were tested for Anti-HBcore (IgM), Anti-HBcore(IgG) and HBsAb. Positive specimens for Anti-HBcore were tested for HBsAg and positive specimens for HBsAg were tested for HBeAg. Data was analyzed by using statistical package of social sciences (SPSS) version (16). Z-test for single proportion was used to compare between the actual prevalence of HBV markers and the expected rates in the absence of risk. P value equal or less than 0.05 was considered statistically significant value. Result: Positive Anti-HBcore (IgG) accounted to 113 (34%), positive HBsAgwas 57 (57%), and positive HBsAb was 7%. The results were significant at 5% confidence level, since all P-values were less than 0.001. There was no positive HBeAgto express high infectivity rate or Anti-HBcore (lgM) to reflect acute infection rate among the respondents. Conclusion: Infection rate of HBV was high, while the related immunity was low among Village Midwives in Khartoum State, Sudan.