Background The prevalence of Helicobacter pylori infection varies in relation to geography, ethnicity and socioeconomic factors. Available data on the prevalence of Helicobacter pylori infection in Uganda are not representative of the general population. We sought to describe the epidemiology of this infection in pregnant women in Uganda to provide background data for a study into the effect of H. pylori infection during pregnancy on the hematological response to iron supplementation. Methods Using a cross-sectional design, H. pylori infection was assessed by the stool antigen test among 447 pregnant women attending antenatal care clinics in Apac, Mbale, Mbarara and Rakai Districts which are in different geographical regions in Uganda, and at Kawempe Health Center which serves a low-income densely populated area in Kampala City. Socio-demographic and household data were collected by face-to-face interviews using a questionnaire. Associations between H. pylori infection and socio-demographic and household characteristics were analyzed using logistic regression. Results The overall prevalence of H. pylori infection was 45.2% but varied by geographical location from 18.2% in Apac District to 60.5% at Kawempe Health Centre. At 18.4%, the Langi ethnic group, who were enrolled exclusively in Apac District, had the lowest prevalence of H. pylori infection while the Gisu had the highest prevalence (58.4%). H. pylori was independently associated with enrollment at clinics not in Apac (adjusted OR = 5.68; 95% CI: 3.02-10.7) and with using water from public wells, boreholes or springs (AOR = 3.20; 95% CI: 1.19-8.61) and from rivers, lakes or streams (AOR = 5.20; 95% CI: 1.58-17.05). Urban residence (AOR = 1.71; 95% CI: 1.13-2.60) and no formal education (AOR = 1.95; 95% CI: 1.03-3.67) were also independently associated with H. pylori infection. Conclusions The unexpected variation in the prevalence of H. pylori infection in Uganda calls for population-based studies in the region and offers an opportunity to study the transmission dynamics of H. pylori infection. The association between H. pylori infection and surface water sources for household use suggests waterborne transmission of H. pylori infection highlighting the need for concerted efforts in environmental health in communities and at the household level.