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The diagnosis of pregnancy in the domestic dog (Canis familiaris) often employs specialized equipment, experienced staff, and the cooperation of the bitch. These procedures can be challenging when the subject is a wild canid, particularly in a field setting. In addition, reproductive hormone assays are unreliable as a diagnostic tool because the estrous profiles of pregnant and pseudopregnant canines are similar. However, research has demonstrated that the hormone relaxin can be detected in maternal blood after embryonic implantation, but remains negligible in non-pregnant females. We investigated the use of relaxin as a diagnostic marker of pregnancy in the coyote (C. latrans). A commercially available canine relaxin enzyme immunoassay (ReproCHEKTM) was used to test plasma collected from 124 female coyotes over four consecutive breeding seasons. Mating activities of the captive females were observed; then peripheral blood samples were collected at intervals throughout pregnancy, as well as after parturition. Results demonstrated that relaxin could be detected in the plasma of pregnant coyotes after 28 days of gestation, and in some cases as early as 23 days, while non-pregnant females and male coyotes consistently tested negative. Relaxin also remained detectable in the plasma of the majority of females tested 10–12 weeks after parturition. This qualitative assay for relaxin proved to be a reliable diagnostic tool for pregnancy in the coyote. In addition, blood sampling was relatively easy, could be accomplished with minimal handling, and did not require sedation or anesthesia.