Abstract Three populations, two from Colombia and one from Brazil, of Rotylenchulus reniformis associated with banana and plantain, were characterized using morphological, morphometric, and molecular methods. Morphometric data from these populations were similar to type and reference populations of R. reniformis . Partial sequences of both D2-D3 rDNA and mitochondrial cytochrome oxidase subunit I (COI) regions had a strong affinity (99% similarity) to previously published sequences of R. reniformis . Phylogenetic analyses (maximum likelihood and Bayesian inference) suggested that the Colombian populations of R. reniformis corresponded to the previously described Type A of the species. This is the definitive first report in Colombia of R. reniformis associated with banana and plantain crops.