Black-tailed prairie dogs (Cynomys ludovicianus) are used as an animal model for research on gallbladder stones and several infectious diseases. A comprehensive, instructive resource regarding the appropriate techniques for venipuncture and collection of nonterminal blood samples in this species has not yet been published. Blood samples (1 mL or larger) were readily obtained from the jugular vein, femoral vein, or cranial vena cava, whereas peripheral sites, such as the cephalic vein, saphenous vein, and tarsal vein, mainly were useful for obtaining smaller volumes. The detailed and illustrated information presented here can aid clinicians and researchers in performing venipuncture, anesthesia, and handling of this species.