Serum samples collected from 20 black rhinoceros (Diceros bicornis) were analyzed for iron values from six different areas in South Africa. In addition, biochemical profiles were performed on individual samples. Comparisons of iron values from free-ranging black rhinoceros and from 28 free-ranging white rhinoceros (Ceratotherium simum) were conducted by location and age. Among the free-ranging black rhinoceros, samples were compared from different regions to a set of samples from black rhinoceros that had been captured and held in bomas. Serum iron levels were not significantly different (P = 0.55) among the three locations with more than one animal (medians 5.57, 5.70, 6.47 ppm), but the median value from the boma group was significantly lower (2.91 ppm; P = 0.042), contrary to previous studies. Similar to reports in captive black rhinos, serum iron levels appeared to show a trend toward increasing values between subadult and adult animals, although differences were not statistically significant among black rhinoceros. Comparison of serum iron levels between free-ranging black and white rhinoceros showed significantly higher median value in black rhinoceros (5.73 ppm) versus white rhinoceros (3.38 ppm, P = 0.001). Other significant differences (P < 0.05) in biochemical values between species included lower median aspartate aminotransferase (37 versus 76.5 U/L), higher copper (1.50 versus 1.34 ppm), higher zinc (1.36 versus 0.37 ppm), lower total protein (8.0 versus 10.35 g/dL), higher gamma glutamyltransferase (13.0 versus 12.5 U/L), and lower globulin (6.6 versus 7.6 g/dL) in black rhinoceros. Further investigations should be conducted to examine the role of age, location, and time in boma confinement on iron values in South African rhinoceros to understand iron metabolism in these species.