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Grasses with desirable traits for agriculture and livestock production are continually being sought. The genus Urochloacomprises many species of good forage potential, although studies have mostly focused on commercial cultivars. Karyotype data can be used to characterize species and generate information for taxonomy and evolutionary studies and contribute to breeding programs. The mode of reproduction is also an essential piece of information to determine breeding strategies. This study aimed to characterize the karyotypes of Urochloa arrecta(Hack. ex T. Dur. & Schniz) O. Morrone & F. Zuloaga and a pentaploid cytotype of U. brizantha(Hochst. ex A. Rich.) R. Webster, including the mapping of ribosomal DNA (rDNA) sites and nuclear DNA content and indicating the mode of reproduction. Slides were prepared using the flame drying technique and were subjected to fluorescent in situ hybridization with 35S and 5S rDNA probes. DNA content was estimated by flow cytometry. The classification of the reproduction mode was based on the amplification of the primers N14 and p779/p780. Urochloa arrectapresented 2C = 2.66 pg of DNA, karyotype formula 2n= 4x= 34 m + 2sm, four chromosomes with 35S rDNA sites, and four other chromosomes with 5S rDNA sites. The absence of amplification products with the primers N14 and p779/p780 provided evidence of sexual reproduction. Urochloa brizanthapresented 2C = 4.52 pg of DNA and karyotype formula 2n= 5x= 45 m. Five chromosomes presented 35S rDNA, and eight other chromosomes displayed 5S rDNA sites. Polymerase chain reaction results indicated that this cytotype has an apomictic mode of reproduction. The organization of karyotypes suggests an allopolyploid origin for both genotypes. Possible applications of the information found are discussed. The marker ASGR–BBML p779/p780 is efficient in discriminating sexual and apomictic Urochloaspp.Cytogenetic data is applied to Urochloabreeding and taxonomic and phylogenetic studies.Karyotype analyses suggest allopolyploid origin in U. arrectaand U. brizantha5x.This is the first study involving karyotype description for U. arrecta.