The East African Mopeia virus (MOPV) is an arenavirus closely related to the highly pathogenic West African Lassa virus, even sharing the same reservoir rodent host Mastomys natalensis. Because MOPV is not known to cause human disease, it offers a unique alternative for studying Lassa virus transmission. We investigated how habitat, population density, and host characteristics are related to MOPV occurrence in M. natalensis populations in Morogoro, Tanzania. In 3 contrasting habitats, 511 M. natalensis individuals were trapped, 12.1% (58/480 tested individuals) of which tested seropositive for antibodies and 8.4% (41/489 tested individuals) for MOPVRNA. Although population densities differ among habitats, density and habitat were not significantly correlated to MOPV-RNA or antibody presence. Antibody presence was not significantly correlated with any host characteristics. In contrast, MOPV-RNA presence was inversely related to weight, age, sexual maturity, and body mass index. The model with body mass index as predictor was the best at predicting infection probability. Thirty-five individuals were exclusively MOPV-RNA positive, 52 were exclusively antibody positive, and 6 were both MOPV-RNA and antibody positive. Interpreting these data using experimental infection results from studies on other arenaviruses, this would mean that these infections were very recent, old, and roughly 1–3 weeks after infection, respectively. The higher RNA prevalence in juveniles implies vertical transmission, or that horizontal transmission occurs mainly in this age group due to lack of immunity, higher susceptibility, and/or higher juvenile contact rates. This study demonstrates the strength of combining information on antibody and RNA presence with host characteristics, and how this information can provide valuable insights into transmission dynamics.