AbstractBACKGROUNDFusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef‐1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non‐toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme‐linked immunosorbent assay (CD‐ELISA) was employed to assess the fumonisin‐producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef‐1α and inter‐simple sequence repeat (ISSR) markers of different Fusarium spp.RESULTSAll 27 isolates of Fusarium spp. were positive for the tef‐1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum‐equiseti complex. The standardized mPCR assay distinguished toxigenic and non‐toxigenic F. verticillioides. Further, mPCR fumonisin‐positive F. verticillioides isolates were also positive by CD‐ELISA. The tef‐1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters.CONCLUSIONThe present method provided rapid and reliable detection of fumonisin‐producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high‐throughput monitoring of major mycotoxin‐producing fungi during the processing steps of food and feed commodities. © 2013 Society of Chemical Industry