Hematology and biochemistry parameters were evaluated in a zoological population of Krefft's river turtles (Emydura macquarii krefftii) to determine the effect of sex and venipuncture site. Blood samples were collected from 17 (9 male, 8 female) healthy adult Krefft's river turtles. Females were reproductively active during the study period. Three venipuncture sites were sampled in each individual, including jugular vein, subcarapacial sinus, and dorsal coccygeal vein. Statistically significant differences were found between venipuncture sites and between sexes. Packed cell volume percentage was significantly lower in samples from the subcarapacial sinus compared to the other sites. Basophil concentration was significantly lower in samples from the subcarapacial sinus compared to the jugular vein. Azurophil, cholesterol, triglycerides, calcium, phosphorus, and magnesium concentrations were significantly higher in females compared to males. Based on the results of this study, the jugular vein is the recommended venipuncture site in Krefft's river turtles. Sex and reproductive status should be assessed at the time of venipuncture when possible and considered in interpretation of hematology and biochemistry results.