Whilst Brazil is the fourth largest cotton producer globally, incidence of ramularia leaf spot (RLS) has decreased yield. In 2017–18 and 2018–19, ca. 300 fungal samples were collected throughout Brazil. Hyphal tip cultures were obtained for amplification of the RNA polymerase II (RPB2), 28S rRNA, the ribosomal DNA internal transcribed spacers (ITS), actin (ACT), elongation factor (EF1-α) and histone H3 (HIS3)genomic regions. Additionally, sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained by nanopore sequencing and theEF1-αregion was selected as a marker for rapid recognition ofRamulariopsisspecies. Clade assignments based on the concatenated-sequence tree were identical to those in tree generated byRPB2-sequences, as well as in anRPB2haplotype network and an ISSR (TGTC)4dendrogram, in identification with species-specific primers and based on morphological comparisons. Out of 267 examined isolates, 252 were identified asRamulariopsis pseudoglycines, indicating this species as the most widespread causal agent of cotton RLS in the Brazilian growing regions. Species-specific primers developed in the study that target theEF1-αgene provide an opportunity for extensive RLS sampling worldwide to study the distribution ofRamulariopsisspecies. Such data will aid breeders and plant pathologists in cotton disease resistance development and fungicide resistance avoidance.