BACKGROUND: Aflatoxin B1 (AFB1) is a secondary toxic metabolite produced by some Aspergillus species, particularly Aspergillus flavus (A. flavus) and A. parasiticus that contaminate feedstuffs. OBJECTIVES: The aim of this study was to evaluate the contamination of the concentrate and corn silage samples to toxigenic fungi and aflatoxin B1 in cattle farms of Yazd province in Iran. METHODS: A total of 80 samples of concentrated feeds and 80 samples of silage feeds were collected from cattle breeding farms of 4 cities in Yazd province in winter and spring seasons (40 samples in each season and one sample from each cattle farm). The samples were cultured on mycological media in order to isolate and determine the amount of the toxigenic fungi. Concurrently, the content of AFB1 was measured in feedstuff samples using ELISA technique. RESULTS: The results indicated that the most frequent fungi isolated were Aspergillus spp. (49.3%), Penicillium spp. (23%), Mucor spp. (22.3%) and Fusarium spp. (4.8%) in winter and Aspergillus spp. (46.9%), Penicillium spp. (21.8%), Mucor spp. (28.5%) and Fusarium spp. (2.8%) in spring from all understudy feedstuffs. The mean of AFB1 in feedstuffs were 0.25 and 0.21 µg/kg in winter and spring, respectively. According to statistical analysis of the results, significant differences were observed between the frequency of Aspergillus isolates and other fungal species (p<0.05) and also between toxigenic fungi, such as Aspergillus, Fusarium and Penicillium, with other isolated fungi (p<0.05). Among toxigenic fungi, significant differences were observed between Aspergillus and Penicillium species, Aspergillus and Fusarium species and Penicillium and Fusarium species (p<0.05). Significant relationship was observed between the amount of toxigenic fungi and AFB1 in feedstuffs (p<0.05). CONCLUSIONS: The results obtained by this study show that rapid and specific detection of aflatoxigenic fungi is essential to ensure the mycological safety of animal feedstuffs.