A comparative map of human chromosome 3 (HSA3) and pig chromosome 13 (SSC13) was constructed using physically assigned pig sequence tagged sites (STSs). Pig STS representing 11 HSA3 genes were developed and 10 pig STS were regionally mapped using a somatic cell hybrid panel (SCHP) to SSC13 with 80–100% concordance. Large-insert probes were obtained by screening a YAC library with primers for each STS. YACs were identified for DRD3, GAP43, PIT1, SI, and SST for fluorescent in situ hybridization (FISH) mapping. Single gene and bicolor FISH with each pairwise combination was used to further define gene order on SSC13. These data confrim chromosome painting results that showed HSA3 probes hybridize to a major portion of pig chromosome 13 and demonstrate extensive gene rearrangements within this conserved synteny group.