The reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant roots and are mainly distributed in tropical and subtropical regions. In this study, we provide morphological and molecular characterisation of six out of ten presently known valid species of Rotylenchulus: R. clavicaudatus, R. leptus, R. macrodoratus, R. macrosoma, R. reniformis and R. sacchari from South Africa, USA, Italy and Spain. Rotylenchulus parvus was only studied morphologically. A new species, R. macrosomoides sp. n., isolated from soil and roots of sugarcane in South Africa, is described. The phylogeny of Rotylenchulus, as inferred from the analyses of D2-D3 of 28S rRNA, ITS rRNA, coxI mtDNA and hsp90 gene sequences, is presented. The study revealed that R. reniformis and R. macrosoma have a sister relationship, but that relationships between other Rotylenchulus species remain unresolved. The phylogenetic analysis also confirmed the hypothesis that this genus originated from the Afrotropical zoogeographical region. Our study revealed that R. reniformis and R. macrosomoides sp. n. have two distinct rRNA gene types and R. macrosoma have three rRNA gene types in their genomes. PCR with species-specific primers was developed for rapid diagnostics of R. reniformis.