The effect of anticoagulant and venipuncture site on hematology and serum chemistries was evaluated in eight captive spiny softshell turtles (Apalone spinifera). Animals were randomly and evenly distributed into two groups. On day 0, blood samples were collected from the dorsal coccygeal vein in turtles from group 1 and from the subcarapacial vein in turtles from group 2. On day 15, the same procedure was repeated, but turtles from group 1 were sampled from the subcarapacial vein and turtles from group 2 from the dorsal coccygeal vein. Hematologic samples were placed in ethylenediaminetetraacetic acid (EDTA) on day 0 and in heparin on day 15. Samples for chemistries were placed in serum separator tubes. All samples in EDTA were hemolyzed. Nonparametric tests were used to assess significant differences in hematologic and biochemical parameters between the two venipuncture sites. Among the biochemical parameters, statistically significant differences were only present for glucose and potassium, with the median values being higher in samples obtained from the subcarapacial vein. Correlation tests did not show any significant correlation between the values obtained from the two venipuncture sites for any of the biochemical parameters evaluated. There was also a general lack of agreement for values obtained from different venipuncture sites. This study concluded that EDTA is not an appropriate anticoagulant for this species, and serum chemistry values from paired samples taken using different venipuncture sites did not have a significant correlation or agreement.