Notopteridae (Teleostei, Osteoglossiformes) represents an old fish lineage with ten currently recognized species distributed in African and Southeastern Asian rivers. Their karyotype structures and diploid numbers remained conserved over long evolutionary periods, since African and Asian lineages diverged approximately 120 Mya. However, a significant genetic diversity was already identified for these species using molecular data. Thus, why the evolutionary relationships within Notopteridae are so diverse at the genomic level but so conserved in terms of their karyotypes? In an attempt to develop a more comprehensive picture of the karyotype and genome evolution in Notopteridae, we performed comparative genomic hybridization (CGH) and cross-species (Zoo-FISH) whole chromosome painting experiments to explore chromosome-scale intergenomic divergence among seven notopterid species, collected in different African and Southeast Asian river basins. CGH demonstrated an advanced stage of sequence divergence among the species and Zoo-FISH experiments showed diffuse and limited homology on inter-generic level, showing a temporal reduction of evolutionarily conserved syntenic regions. The sharing of a conserved chromosomal region revealed by Zoo-FISH in these species provides perspectives that several other homologous syntenic regions have remained conserved among their genomes despite long temporal isolation. In summary, Notopteridae is an interesting model for tracking the chromosome evolution as it is (i) ancestral vertebrate group with Gondwanan distribution and (ii) an example of animal group exhibiting karyotype stasis. The present study brings new insights into degree of genome divergence vs. conservation at chromosomal and sub-chromosomal level in representative sampling of this group.