Release of granzymes and perforin from the cytolytic granules of SIV-specific CD8 T cells is a critically important effector mechanism facilitating the elimination of SIV-infected cells. We sequenced granzyme A, B, and K and perforin in pigtail macaques and defined polymorphisms between humans, rhesus macaques, and pigtail macaques. The pigtail macaque sequences were similar to the corresponding rhesus sequences at the mRNA and protein level and (0.4-1.1% sequence differences) but substantially different from human sequences (3.8-8.1% sequence differences). We used this sequence information to develop multiplex PCR assays to detect these genes. We also successfully studied the release of perforin and granzyme B from deregulating SIV-specific CD8 T cells by flow cytometry. These sequences and tools enable further study of the cytolytic control of SIV in pigtail macaques.