Bilateral anterior amelia (BAA) is the congenital absence of thoracic limbs and has been reported in the Chihuahua as an autosomal recessive disorder. In some cases, the digits of the pelvic limbs can be variably affected, but otherwise, the pelvic limbs are generally spared. A GWAS performed with nine BAA affected Chihuahuas identified a significant association on chromosome 13, and homozygosity mapping delineated a 2.1 Mb chromosomal region containing the RSPO2 gene. Loss of function variants of RSPO2 in humans and cattle has been associated with the absence of all limbs. Six affected Chihuahuas were whole genome sequenced (WGS) and aligned to the CanFam4 assembly. SNVs, small indels, and structural variants within the critical interval that fitted a recessive model were investigated. Three SNVs (NC_049234.1:g.8891861C > T; NC_049234.1:g.8974204C > T and NC_049234.1:g.9789424G > A) were homozygous in five cases and absent from 3,418 genetically diverse control genome sequences, except for one Small Poodle that was heterozygous. One SNV resided in RSPO2's second intron, while the two others were intergenic. The three candidate variants were genotyped in 7 additional cases and 100 control Chihuahuas. Twelve of 13 cases were homozygous for the mutant allele, and one case was heterozygous. Controls were either homozygous for the reference allele (97%) or heterozygous (3%). Our data should facilitate genetic testing of Chihuahuas to prevent the unintentional production of BAA affected dogs. Moreover, the identification of these variants enhances understanding of RSPO2 gene function in limb development.