Osteoglossiformes is one of the most ancient (Early Triassic) groups of freshwater teleosts and has at least one representative on each continent of the Southern Hemisphere, with the exception of Antarctica. Among its six described families, the Gymnarchidae occurs in many African rivers, including the river Nile, where Gymnarchus niloticus is its only representative. In this study we provide, for the first time, a comprehensive cytogenetic analysis for G. niloticus, which includes definition of the standard karyotype, C-banding, repetitive DNA mapping and comparative genomic hybridization, in order to investigate its chromosomal patterns and provide new data for comparative analyses with other Osteoglossiformes species. The 2n was 34 (26 metacentric + 8 submetacentric) in both sexes, and C-positive heterochromatic bands were detected in the centromeric/pericentromeric regions of all chromosomes. The simultaneous detection of 18S and 5S ribosomal DNAs showed simple and non-syntenical locations for such sequences, and a Chromomycin A3-positive band was observed, which is co-localized within the 18S ribosomal RNA gene sequences. Fluorescence in situ hybridization using U2 small nuclear RNA, Rex6 and some microssatelites as probes showed a scattered distribution among chromosomes. Data from comparative genomic hybridization experiments with some other African Osteoglossiformes species showed that all these species have an advanced stage of sequence divergence. The results highlight the evolutionary diversification of osteoglossiform fishes.