Canine Sry-negative XX sex reversal is a disorder of gonadal development wherein individuals having a female karyotype develop testes or ovotestes. In this study, linkage mapping was undertaken in a pedigree derived from one proven carrier American cocker spaniel founder male and beagle females. All affected dogs in the analysis were XX true hermaphrodites and confirmed to be Sry negative by polymerase chain reaction. A genome-wide linkage screen conducted using 245 microsatellite markers revealed highest LOD score of 3.4 (marker CPH9) on CFA29. Fine mapping with additional microsatellites in the region containing CPH9 localized the Sry-negative XX sex reversal locus to a 5.4-Mb candidate region between markers CPH9 and FH3003 (LOD score 3.15). Insignificant LOD scores were found at genome-wide screen or fine mapping markers that were within 10 Mb of 45 potential candidate genes reported to have a role in mammalian sex determination or differentiation. Together, these results suggest that a novel locus on CFA29 may be responsible for sex reversal in this pedigree.