Fluctuations in seasonal patterns of horse fly populations were examined in rainforests of tropical South America, where the climate is seasonal. These patterns were evaluated with robust analytical models rather than identifying the main factors that influenced the fluctuations. We examined the seasonality of populations of horse flies in fields and lowland areas of the Pampa biome of southern Brazil with generalized linear models. We also investigated the diversity of these flies and the sampling effort of Malaise traps in this biome over two years. All of the 29 species had clear seasonality with regard to occurrence and abundance, but only seven species were identified as being influenced by temperature and humidity. The sampling was sufficient and the estimated diversity was 10% more than observed. Seasonal trends were synchronized across species and the populations were most abundant between September and March and nearly zero in other months. While previous studies demonstrated that seasonal patterns in population fluctuations are correlated with climatic conditions in horse fly assemblages in South America rainforests, we show a clear effect of each factor on richness and abundance and the seasonality in the prevalence of horse fly assemblages in localities of the Pampa biome.