Potamotrygonidae is a family of freshwater stingray endemic in South America. We determined the hematological and serum biochemistry parameters of Potamotrygon motoro and Potamotrygon falkneri from Parana Basin and Potamotrygon orbignyi and Potamotrygon scobina from the Amazon Basin. Blood was collected from 55 specimens of Potamotrygon sp., and these parameters were evaluated: red blood cell count, hematocrit percent, hemoglobin concentration, leucocytes count, trombocytes count, total protein concentration, albumin, uric acid, urea, creatinine, triglycerides, cholesterol, high-density lipoprotein HDL, calcium, phosphorus, calcium/phosphorus relationship, creatine kinase CK, gamma-glutamyl transpeptidase GGT, Aspartate transaminase AST, alanine transaminase ALT, globulin, albumin/globulin relationship, alkaline phosphatase AP, sodium, chloride, potassium, triiodothyronine T3, and thyroxine T4 hormones. The hematological indices were similar for all species, except for levels of heterophils in P. orbignyi and P. scobina that were higher. Calcium, phosphorus, calcium/phosphorus relationship, sodium, potassium, chloride, AST, ALT, GGT, CK, albumin/globulin relationship, uric acid, creatinine, urea, triglycerides, HDL, T3, T4 levels had no significant difference between the species. These results suggest that there is low influence of habitat and feeding habits. AP, total protein, albumin, globulin, glucose, and total cholesterol had significant differences between the studied species. These results support the theory that stingrays migrated from the Atlantic Ocean, adapting to different conditions over time and placed themselves geographically distant from each other.