Evolutionarily related species often share a common order of genes along homeologous chromosomes. Here we report the collinearity disruption of genes located on homeologous chromosome 4 in Allium species. Ultra-sensitive fluorescence in situ hybridization with tyramide signal amplification (tyr-FISH) allowed the visualization of the alliinase multigene family, chalcon synthase gene and EST markers on Allium cepa and Allium fistulosum chromosomes. In A. cepa, bulb alliinase, root alliinase (ALL1) and chalcon synthase (CHS-B) genes were located in the long arm but EST markers (API18 and ACM082) were located in the short arm. In A. fistulosum, all the visualized genes and markers were located in the short arm. Moreover, root alliinase genes (ALL1 and AOB249) showed contrast patterns in number of loci. We suppose that the altered order of the genes/markers is the result of a large pericentric inversion. To get insight into the evolution of the chromosome rearrangement, we mapped the bulb alliinase gene in phylogenetically close and distant species. In the taxonomic clade including A. fistulosum, A. altaicum, A. oschaninii and A. pskemense and in phylogenetically distant species A. roylei and A. nutans, the bulb alliinase gene was located on the short arm of chromosome 4 while, in A. cepa and A. schoenoprasum, the bulb alliinase gene was located on the long arm of chromosome 4. These results have encouraging implications for the further tracing of inverted regions in meiosis of interspecific hybrids and studding chromosome evolution. Also, this finding may have a practical benefit as closely related species are actively used for improving onion crop stock.