AbstractThe brown‐throated sloth, Bradypus variegatus, is a common species endemic to South and Central America. Nonetheless, maintaining these animals in captivity can be challenging, and very few institutions manage to do so. The São Paulo Zoological Park Foundation is in a remnant of the Atlantic rainforest in the middle of São Paulo, the largest city in Brazil. This forest fragment has a population of B. variegatus that is geographically isolated and yet to be studied. Assessing the health status of individuals remains difficult due to the lack of reference intervals (RIs) for hematologic and biochemical variables for this species. We aimed to establish hematologic and biochemical RIs in a population of wild B. variegatus living in the largest remnant of Atlantic rainforest in São Paulo city, Brazil. Blood samples from 25 individuals of wild B. variegatus were collected and analyzed for 20 hematologic and 21 biochemical variables, using standard laboratory techniques. Each variable was statistically analyzed according to the American Society for Veterinary Clinical Pathology guidelines. The results obtained for each variable were statically analyzed, making it possible to obtain descriptive statistics for all hematologic and biochemical variables. RIs were determined for 16 hematologic variables. During the microscopic analysis, we observed anisocytosis, polychromatophils, Howell‐Jolly bodies, macroplatelets, and reactive lymphocytes. The RIs and descriptive statistics described here establish important baseline numbers that could be essential for the management and treatment of both captive and wild B. variegatus sloths.