The prevalence of human T-cell lymphotropic virus (HTLV-1/2) infection among puerperae in the State of Mato Grosso, Brazil, is unknown. Through this cross-sectional study, the prevalence of HTLV-1/2 infection among puerperae attended at three public maternity hospitals in Cuiabá, State of Mato Grosso, was defined. Between April and September 2006, 3,831 deliveries took place and 2,965 puerperae underwent serological tests for HTLV-1/2: enzyme-linked immunosorbent assay (ELISA) and western blot. The mean age of the women studied was 23.9 years. The prevalence of HTLV-1/2 was 0.2%, i.e. similar to the prevalence observed in the general population of many developed centers in Brazil. This finding of low prevalence suggests that there is still no justification for introducing public health interventions for the population of pregnant women in our setting, to reduce the vertical transmission of HTLV-1/2.