Urochloa P. Beauv. [syn. Brachiaria (Trin.) Griseb.] belongs to the family Poaceae and comprises species presenting different ploidy levels and modes of reproduction. Specific studies on genomic constitution and relationship within the genus Urochloa are limited, despite the great economic and agronomic importance of forage species and the high frequency of polyploids. Therefore, molecular cytogenetic analyses can enhance the knowledge about genomes and their differentiation, which allows making inferences about phylogenetic and genomic relationships. The current study aimed to investigate the genomic relationships between Urochloa ruziziensis (R. Germ. & Evrard) Crins (syn. Brachiaria ruziziensis Germain & Evrard.) cultivar ‘Kennedy’, Urochloa decumbens (Stapf) R. D. Webster [syn. Brachiaria decumbens (Stapf)] cultivar ‘Basilisk’, Urochloa brizantha (Hochst. ex. A. Rich.) R. D. Webster [syn. Brachiaria brizantha (A. Rich.) Stapf] cultivar ‘Marandu’, and their interspecific hybrids by means of genomic in situ hybridization, fluorescent in situ hybridization, and nuclear genome quantification by flow cytometry. We proposed the genomic constitution of U. ruziziensis, U. decumbens, and U. brizantha, such as B2B2, B1B1B2B2, and BBB1B1, respectively. Genomes B, B1, and B2 were considered to be homoeologous, but there was less affinity between genomes B and B2.