The purpose of this study was to develop a molecular method for detecting polled intersex syndrome (PIS) genetic deficiency gene in dairy goat. Three pairs of primers, PIS-, PIS+, and NEI were designed based on PIS gene sequence (AF404302) to identify the PIS genetic deficiency genotype. For the normal phenotype, the fragments of 141 and 300 bp were obtained for the genotype PIS-PIS-, and 141, 449, and 300 bp for the genotype PIS-PIS+. For the PIS goat with the genotype PIS+PIS+, 449 and 300 bp were obtained. Two hundred and twenty-four dairy goats in one population were tested based on this method. The results showed that there were 150 PIS-PIS+, 70 PIS -PIS-, and 4 PIS+PIS+. The genotype frequency of PIS-PIS+ was 66.9%, and the gene frequency of PIS+ was 35.3% in the population. Therefore, the frequency of PIS offspring was over 12%. This study developed a method to detect PIS genetic deficiency dairy goat. The method could identify buck genotype accurately to avoid the occurrence of PIS genetic deficiency. The ease and accuracy show a strong potential of the method for use in marker assisted selection of dairy goats and healthy development of dairy goat industry.