From June 2014 to June 2015, capillary tube collections of blood were obtained concurrently with ear clips of trapped free-ranging, globally vulnerable New England cottontails (NEC; Sylvilagus transitionalis) and eastern cottontail rabbits (EC; Sylvilagus floridanus) in the Hudson Valley region of New York, United States. Species identification (NEC, EC) and sex (NEC) were determined genetically using a mitochondrial DNA assay and Y chromosome marker, respectively. Hematocrit values were obtained using a microhematocrit centrifuge. We provide the reference values 35.15–49.55 (2.5 and 97.5 percentiles) and 90% confidence intervals (CI) [lower: 33.00, 36.08; upper: 46.95, 51.00], for hematocrit of NEC. The mean hematocrit for NEC was 42.35% (SE = 0.58, n = 47) and a comparative contemporaneous mean in the same area for EC [39.96 (SE = 0.81, n = 26)], which was significantly different from NEC (P = 0.02). There was a significant sex difference for NEC [male: 43.99 (SE = 1.02, n = 28); female: 39.92 (SE = 0.78, n = 19), P < 0.0001], though not for EC.