Heavy metals are among the most important environmental pollutants which accumulate in various organs and are associated with several toxic effects. This study was performed to determine the status of heavy metals in river buffaloes in Khuzestan province, Iran, and its relationship with hematologic and serum biochemical parameters. A total of 103 apparently healthy buffaloes were sampled from the region. The concentration of heavy metals, including lead (Pb), mercury (Hg), and cadmium (Cd), was determined in serum samples by atomic spectroscopy. In addition, complete blood counts and serum biochemical profiles were assessed. The serum concentration of Cd, Pb, and Hg in the sampled buffaloes, as mean±standard error, were 0.55±0.01, 6.51±0.10, and 6.28±0.09 µg/l, respectively, which are within the permissible serum levels in the livestock. Serum Cd and Hg levels showed no significant relationship with hematologic or biochemical analytes. However, there were significant negative correlations between Pb levels and phosphorus, magnesium, sodium, as well as potassium concentrations, while serum iron was positively correlated with lead (P<0.05). In addition, there was a significant positive correlation between Hg level and serum aspartate aminotransferase activity (P<0.05). Despite the fact that river buffaloes in Khuzestan spend a long time daily in the Karun River with high industrial pollution, no serum evidence of heavy metal toxicity was found in these animals. It can be suggested that river buffaloes in Khuzestan seem to be resistant to the environmental pollution caused by heavy metals. However, further studies are required to confirm this issue and identify its possible explanations.