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Arowanas (Osteoglossinae) are charismatic freshwater fishes with six species and two genera (<i>Osteoglossum</i> and <i>Scleropages</i>) 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 2<i>n</i> = 56 and 54 in <i>O. bicirrhosum</i> and <i>O. ferreirai</i>, respectively, and 2<i>n</i> = 50 in <i>S. formosus,</i> while <i>S. jardinii</i> and <i>S. leichardti</i> presented 2<i>n</i> = 48 and 44, respectively. A time-calibrated phylogenetic tree revealed that <i>Osteoglossum</i> and <i>Scleropages</i> divergence occurred approximately 50 million years ago (MYA), at the time of the final separation of Australia and South America (with Antarctica). Asian <i>S. formosus</i> and Australian <i>Scleropages</i> 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.