Blood samples from 172 captive and 40 wild, healthy, juvenile and adult, ploughshare tortoises (Astrochelys yniphora) were analyzed to determine hematological and biochemical reference intervals. Hematological analytes included packed cell volume (PCV), white blood cell count (WBC), and WBC differential estimates. Biochemical analysis included total protein measured by photometry (TP) and by refractometry (TPr), albumin (ALB), creatine kinase (CK), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), bile acids (BA), calcium (Ca), phosphorus (P), urea (UREA), and uric acid (UA). The jugular vein was identified as the preferred venipuncture site as subcarapacial vein venipuncture resulted in regular hemodilution. In due consideration of small sample sizes in some of the groups studied, adult tortoises had significantly higher plasma GLDH activity and TPr, TP, ALB, BA, and UREA concentrations and significantly lower AST activity and P concentration than juveniles. Captivity had a significant influence in some reference intervals, with captive adults presenting significantly higher WBC, and estimated counts of all white cell types as well as UREA and TPr than wild counterparts. Captive juveniles also showed significantly higher estimated monocyte and lower estimated eosinophil and basophil counts. Although these differences most likely reflect local environmental or dietary differences, without representing pathology or a deviation from the normal, they question the applicability of reference values from captive animals to wild animals and vice versa. Significant sex differences were only observed for PCV and UA. The reported reference intervals may serve as benchmarks for clinical assessment and conservation of this critically endangered species.