Rattlesnakes are heterothermic and depend on external sources of heat to regulate its body temperature and other physiological processes. These heat sources vary according to the season and this variation difficult the interpretation of test results.The aim of this study is to determine the changes that occur seasonally in the biochemical profile of rattlesnakes and check for variations resulting from the sex of animals in captivity. For determining the concentrations of plasma biochemical parameters of rattlesnakes in captivity, 60 adult snakes were used, 30 males and 30 females. The blood of each snake was collected in summer and winter, and statistical analysis were made to verify it there was seasonal and sexual influence. The values obtained in this study are similar to those previously reported for snakes, and the differences observed are probably due to the difference between species, climate, season and methodology used. The results indicated that there are seasonal influences in the concentrations of total protein, albumin, globulin, uric acid, creatinine, urea, HDL-C, total calcium, magnesium, ALT, GGT, ALP, CK and LDH. Also differences were found between males and females concentrations of cholesterol, HDL-C, VLDL-C, triglycerides, total calcium, ionized calcium and FAL.These results reinforce the importance of considering the seasonal and sexual influence in the interpretation of biochemical parameters of rattlesnakes.