Ramularia leaf spot is a disease of major importance on cotton fields in Brazil due to its effects on yield and cotton fiber quality. Two Ramulariopsis (syn. Ramularia) species, R. gossypii and R. pseudoglycines, are reported as the causal agents of this disease, but it is unknown which species is the most prevalent in Brazilian cotton fields. The goal of this work was to determine the most frequent species occurring on field samples from a molecular monitoring program that sampled from all cotton-growing regions in Brazil from 2017 to 2020 seasons. We also used molecular tools for genotyping a region of the CYTB gene for all sampled isolates. Sequencing of an ITS-rDNA region was used for Ramulariopsis species determination, and a DNA fragment from the CYTB gene was amplified, sequenced, and analyzed for all 165 isolates. The ITS-rDNA sequencing confirmed that all isolates belong to Ramulariopsis, and most notably, all the single nucleotide polymorphisms observed in this region, are of the R. pseudoglycines species for all 165 isolates; thus all analyzed isolates were assigned to this species. The analysis of the CYTB gene fragment sequenced showed the presence of the G143A substitution, and absence of G137R substitution, in all 165 isolates.