Spondias L. comprises at least nine Neotropical species, including the widely cultivated S. monbim and S. tuberosa. Umbu-cajá, a putative hybrid between these two species, is also grown. In this paper, the karyotypes of five Spondias species and Umbu-cajá were analysed for evidence of this hybridization. Chromosome banding with chromomycin A3 and the distribution of 5S and 45S rDNA sites were used to characterize the plants, also genomic in situ hybridization using nuclear DNA from both putative parents and the hybrid as probes. All material presented the same chromosome number (2n = 32) and morphology, but differed in the number and distribution of bands. Spondias monbim and S. tuberosa, the supposed relatives of Umbu-cajá, displayed similar banding patterns, with five to six chromosome pairs having terminal bands, whereas Umbu-cajá exhibited bands on both members of nine chromosome pairs. The three other species, S. venulosa, S. cytherea and S. purpurea, showed less closely related karyotypes, with bands in 12-18 chromosome pairs. In situ hybridization with 5S and 45S rDNA probes revealed one site of each probe per haploid chromosome complement in all material. However, in S. tuberosa, the location of 5S rDNA was different from the other species and found no counterpart in Umbu-cajá. Several tests with total DNA from S. mombin and S. tuberosa against metaphase chromosomes of Umbu-cajá failed to differentiate the individual genomes in the hybrid. From the chromosome banding and the distribution of rDNA sites, as well as from the genomic in situ hybridization, it seems clear that Umbu-cajá is related closely to S. monbim and S. tuberosa, but it is karyotypically homozygous and distinct from theses other species. Karyotypically, the three other investigated species were related less closely to Umbu-cajá.