Although Tragulidae, as the basal family in Ruminantia phylogenetic tree, is the key taxon for understanding the early chromosome evolution of extant ruminants, comparative molecular cytogenetic data on the tragulids are scarce. Here, we present the first genome-wide comparative map of the Java mouse deer (Tragulus javanicus, Tragulidae) revealed by chromosome painting with human and dromedary probes. Together with the published comparative maps of major representative cetartiodactyl species established with the same set of probes, our results allowed us to reconstruct a 2n = 48 Ruminantia ancestral karyotype, which is similar to the cetartiodactyl ancestral karyotype. The karyotype evolution of T. javanicus has involved multiple rearrangements, most of which appear to be apomorphic and have not found in karyotype evolution of pecoran species (i.e., Ruminantia excluding Tragulidae). The rate of chromosome evolution of the mouse deer was rather low-0.4 R/Ma, while the estimated tempo of chromosome changes on the lineages leading from Cetartiodactyla ancestor to Ruminantia and from Ruminantia to Pecora were roughly the same (about 1.2 R/Ma).