The aims of the study were to outline the sequence of events that gave rise to the vertebrate insulin-relaxin gene family and the chromosomal regions in which they reside. We analyzed the gene content surrounding the human insulin/relaxin genes with respect to what family they belonged to and if the duplication history of investigated families parallels the evolution of the insulin-relaxin family members. Markov Clustering and phylogenetic analysis were used to determine family identity. More than 15% of the genes belonged to families that have paralogs in the regions, defining two sets of quadruplicate paralogy regions. Thereby, the localization of insulin/relaxin genes in humans is in accordance with those regions on human chromosomes 1, 11, 12, 19q (insulin/insulin-like growth factors) and 1, 6p/15q, 9/5, 19p (insulin-like factors/relaxins) were formed during two genome duplications. We compared the human genome with that of Ciona intestinalis, a species that split from the vertebrate lineage before the two suggested genome duplications. Two insulin-like orthologs were discovered in addition to the already described Ci-insulin gene. Conserved synteny between the Ciona regions hosting the insulin-like genes and the two sets of human paralogons implies their common origin. Linkage of the two human paralogons, as seen in human chromosome 1, as well as the two regions hosting the Ciona insulin-like genes suggests that a segmental duplication gave rise to the region prior to the genome doublings. Thus, preserved gene content provides support that genome duplication(s) in addition to segmental and single-gene duplications shaped the genomes of extant vertebrates.