The development of a detailed genome map for the domestic dog (Canis familiaris, CFA) is a prerequisite for the continued use of this species as a model system for the study of inherited traits. We present an integrated cytogenetic, radiation-hybrid, and comparative map of dog Chromosome (Chr) 5 (CFA 5). The map comprises 14 gene markers, selected from loci previously mapped within the corresponding evolutionarily conserved chromosome segments (ECCS) of the human genome. Large-insert clones representing each marker were first isolated and mapped by fluorescence in situ hybridization (FISH) analysis to determine their subchromosomal localization on CFA 5. Thirteen gene markers were subsequently mapped by using a commercially available whole genome radiation hybrid (WG-RH) panel for the dog. Nine anonymous markers were also assigned to CFA 5 by both FISH and WG-RH analysis. The 22 markers formed six RH-linkage groups, spanning each of the four ECCS comprising this 99 megabase chromosome. All cytogenetic, WG-RH, and comparative mapping data were in agreement and were combined to determine both the most likely locus order within each linkage group, and also the gross relative orientation of the corresponding ECCS. This study provides a resource for the transfer of information from the human transcript map to that of the dog, and extends existing data regarding the structural relationships between CFA 5 and its evolutionary counterparts within the human genome.