Species of Oxalis subgenus Thamnoxys show a wide variation in karyotype and have a confused taxonomy. Oxalis psoraleoides, 2 n = 12, and O. rhombeo-ovata, 2 n = 14, stand out due to their uncommonly large chromosomes. They are placed, respectively, in sections Psoraleoideae and Polymorphae, suggesting the occurrence of two independent events of increase in genome size or perhaps an artificial taxonomic grouping. A cytogenetic analysis of these species and of five others in section Polymorphae was performed with fluorochrome banding, in situ hybridization of 5S and 35S rDNA probes and DNA content estimations, and a molecular approach was used to determine their phylogenetic relationships. Our results revealed that O. rhombeo-ovata and O. psoraleoides share the same number of chromosome arms, a single pair of 5S rDNA sequences and chromomycin A3 positive bands (CMA+) bands on the long arms. On the other hand, O. rhombeo-ovata has only half the genome size of O. psoraleoides, a putative centric fission, an extra heterochromatic band and 35S rDNA sequences spread on the short arm of all chromosomes. Remaining species of O. section Polymorphae had 2 n = 10, a smaller genome size and a distinct karyotype formula. The phylogenetic analyses retrieved O. psoraleoides and O. rhombeo-ovata as closely related species, supporting the cytogenetic results and suggesting they should be placed together in O. section Psoraleoideae. A taxonomic revision and a well-represented molecular phylogenetic analysis of these sections are needed to better understand their evolutionary relationship and karyotype evolution. [ABSTRACT FROM AUTHOR]