Seasonal fluctuations of the Simulium damnosum Theobald complex and the prevalence of Onchocerca volvulus Bickel in blackflies caught in river systems bordering the Nigeria–Benin border were assessed for their direct impact on the epidemiology of onchocerciasis in South-western Nigeria. Entomological evaluation and heteroduplex assay (HDA) techniques were performed on flies caught in the eight capture points in the Ogun and Yewa river systems between October and December 2007, July and December 2008 and May and December 2009. A total of 5789 blackflies were caught on human bait, of which 727 (12.6%) flies were captured in 2007, 1723 (29.8%) in 2008 and 3339 (57.6%) in 2009. The majority of flies caught during the study were forest flies representing 90.3% of the total catch while savanna flies constituted 9.7%. Proportions of parous to nulliparous flies were low in all the catching points (31.1 and 68.9%, respectively). Of the 5789 flies dissected, 11 (0.2%) flies were infected with Onchocerca parasites with nine of the infected flies having L3 head parasites. The HDA results revealed that the Beffa form of S. soubrense was the dominant cytospecies present (87.1%) in all the capture sites when compared with 12.9% of S. damnosum s.s. The low level of infectivity of flies may therefore indicate a low transmission level of onchocerciasis in the communities along the Ogun and Yewa river systems. However, there is a need for constant surveillance on species composition and fly infectivity in the river systems along the borders of Nigeria–Benin Republic.