Characterization of aflatoxin-producing fungi on postharvest quality of stored Sorghum (Sorghum bicolor L.) grains in Makurdi, Benue State, was carried out. Sorghum samples were collected from storehouses in Wurukum, Yaikyo market and Northbank, respectively, using the sampling probe method. The samples were taken to Botany Laboratory of Rev. Fr. Moses Orshio Adasu University for isolation of aflatoxin-producing fungi, while samples were sent to the Institute for Agricultural Research, Zaria, Kaduna State, for aflatoxin analysis. Fungal isolation from Sorghum seeds were carried out using the Standard Blotter Method, while aflatoxin analysis was done using the Enzyme-Linked Immunosorbent Assay (ELISA) technique. The collected data were subjected to statistical analysis using the chi-square (χ²) test, t-test, and analysis of variance (ANOVA). Where significant differences occurred, mean values were compared using Fisher’s least significant difference (LSD) test at the 5% probability level. The mycological assessment of Sorghum grains resulted in the identification of three fungal genera/species, namely Aspergillus niger, Penicillium sp., and Aspergillus flavus. Statistical analysis indicated that the average fungal load of Sorghum grains did not differ significantly among the stores from which the samples were collected. Among the fungi detected, Aspergillus flavus showed the greatest frequency of occurrence, accounting for 8 isolates (50.0%), followed by Penicillium sp. with 7 isolates (36.8%). Aspergillus niger was recorded at the lowest frequency, comprising 4 isolates (21.1%). Sorghum grains from a storehouse in Northbank had higher fungi occurrence, 7 (36.8%), followed by those in Wurukum and Yaikyo market, each with 6 (31.6%), respectively. There was no significant relationship in fungal occurrence based on location (χ2= 2.45; df= 4; P= 0.65). There was no significant difference in seed germination. Grains from the storehouse in Yaikyo market and Northbank had higher germination, each with (7.33), and the least was that from Wurukum (7.00). Sorghum grains from the storehouse in Yaikyo market had a higher vigour index (820.00), followed by grains from the storehouse in Wurukum (743.80) and Northbank (526.70), respectively. Aflatoxin B1 was identified in Sorghum samples collected from the different storehouses. Aflatoxin level in Sorghum grains obtained from Northbank (6.85) µg/kg was significantly higher compared with aflatoxin levels in Sorghum from Yaikyo market (1.34) µg/kg and Wurukum (0.00) µg/kg, respectively. Aflatoxin B1 was detected in all Sorghum samples collected from the different storehouses in Makurdi. The levels of aflatoxin in Sorghum from Yaikyo market and Northbank stores were below the permissible limit by the Standard Organisation of Nigeria, but the level of aflatoxin in Sorghum from the store in Northbank exceeded the permissible limit by the European Union. Farmers within and outside Makurdi, Benue State, should source high-quality Sorghum grains from non-aflatoxin-contaminated stores for planting.