Extensive use of the organophosphate diazinon has led to its accumulation in aquatic environments and negative effects on fish health. Most studies focus on the effects of short term exposure to high levels of organophosphate pesticides. This research was conducted to assess the effect of chronic sub-lethal exposure to 0, 0.01, and 0.1 mg/L diazinon for 15 and 30 days on blood parameters and histopathological damage in alligator gar, Atractosteus spatula. Fish exposed to either concentration were motionless, produced excess mucous, had lighter skin color, and had skin lesions. Blood indices of red blood cells, leukocytes and hematocrit values significantly decreased but there was no significant change in mean cell volume. Hemoglobin values significantly increased in fish exposed to the low dose for 15 days, but significantly decreased for the other exposure doses and times. On the contrary, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration values significantly increased in fish exposed to both concentrations and duration times. Leukocyte differentials showed significant decreases in lymphocytes with significant increases of monocytes in fish exposed to the high dose in both exposure times. The most frequent biomarker for exposure to organophosphate pesticides is the inhibition of cholinesterase (ChE). Diazinon significantly reduced plasma ChE activity in a dose-dependent manner, with 62% and 72% in the low and high concentrations for 30 days, respectively. Plasma ChE could be determined from peripheral blood samples and did not require sacrifice of the fish. Fish exposed to either diazinon concentration or duration time developed histopathological changes in skin, gills, liver, and kidney tissues. The skin lesions were on the head and body, and progressed into deep ulcerations. The histopathological changes in the liver included hepatic vacuoles, swollen hepatocytes, steatosis, aggregation of macrophages, necrosis, and hepatic fibrosis. Also, exposed fish demonstrated vacuolar degeneration in the hematopoietic tissues of the kidney. Gills showed epithelial hyperplasia in the secondary lamellae. In conclusion, long term exposure to sub-lethal concentrations of diazinon induced significant changes in hematological indices and histopathological alteration in various tissues. Plasma ChE can be used to monitor diazinon exposure in wild gar populations.