This study aimed to determine the aflatoxin B1 (AFB1) levels in feed-stuff samples. For this purpose, 90 animal feed samples including barley, wheat bran,wheat pulp, canolameal, safflowermeal, cottonseedmeal and sunflower meal were randomly obtained from retail stores in Mashhad-Iran and analyzed by both enzyme linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) techniques. Results showed presence of AFB1 in 33 (36.67%) and 10 (11.11%) samples analyzed by HPLC and ELISAtechniques, respectively. The average±SD concentration ofAFB1 in the sampleswas 2.21±2.25 and 10.76±0.86 µg/kg for HPLC and ELISAtechniques, respectively. The range of contamination reported 0.34- 5.81 and 9.5-12.6 µg/kg for the mentioned techniques respectively. ELISA showed reliability and a high correlation with HPLC of 0.93 indicating its potential for aflatoxin screening in animal feed samples. However, sensitivity and specificity of HPLC was higher than ELISA method.