PREMISE OF THE STUDY: Polyploidy has been long recognized as an important force in plant evolution. Previous studies had suggested widespread occurrence of polyploidy and the allopolyploid origin of several species in the diverse Neotropical genus Lachemilla (Rosaceae). Nonetheless, this evidence has relied mostly on patterns of cytonuclear discordance, and direct evidence from nuclear allelic markers is still needed. METHODS: Here we used PCR target enrichment in combination with high throughput sequencing to obtain multiple copies of the nuclear ribosomal (nr) DNA cistron and 45 regions of the plastid genome (cpDNA) from 219 accessions representing 48 species of Lachemilla, and explore the allopolyploid origin of species in this group. KEY RESULTS: We were able to identify multiple nrDNA ribotypes and establish clear evidence of allopolyploidy in 33 species of Lachemilla, showing that this condition is common and widespread in the genus. Additionally, we found evidence for three autopolyploid species. We also established multiple, independent origins of several allopolyploid species. Finally, based solely on the cpDNA phylogeny, we identified that the monotypic genus Farinopsis Chrtek & Soják is the sister group of Lachemilla and allied genera within subtribe Fragariinae. CONCLUSIONS: Our study demonstrates the utility of the nuclear ribosomal DNA cistron to detect allopolyploidy when concerted evolution of this region is not complete. Additionally, with a robust chloroplast phylogeny in place, the direction of hybridization events can be established, and multiple, independent origins of allopolyploid species can be identified.