Arowanas (Osteoglossinae) are charismatic freshwater fishes with six species and two genera (Osteoglossum and Scleropages) distributed in South America, Asia, and Australia. In an attempt to provide a better assessment of the processes shaping their evolution, we employed a set of cytogenetic and genomic approaches, including i) molecular cytogenetic analyses using C- and CMA3/DAPI staining, repetitive DNA mapping, comparative genomic hybridization (CGH), and Zoo-FISH, along with ii) the genotypic analyses of single nucleotide polymorphisms (SNPs) generated by diversity array technology sequencing (DArTseq). We observed diploid chromosome numbers of 2n = 56 and 54 in O. bicirrhosum and O. ferreirai, respectively, and 2n = 50 in S. formosus, while S. jardinii and S. leichardti presented 2n = 48 and 44, respectively. A time-calibrated phylogenetic tree revealed that Osteoglossum and Scleropages divergence occurred approximately 50 million years ago (MYA), at the time of the final separation of Australia and South America (with Antarctica). Asian S. formosus and Australian Scleropages diverged about 35.5 MYA, substantially after the latest terrestrial connection between Australia and Southeast Asia through the Indian plate movement. Our combined data provided a comprehensive perspective of the cytogenomic diversity and evolution of arowana species on a timescale.