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Fusarium proliferatum is a globally distributed fungal pathogen that affects a range of crop hosts and is one of the main producers of mycotoxins, such as fumonisins, in foods. Specific PCR primers are commonly used for detection and identification of this pathogen. The aim of this study was to validate previously published F. proliferatum‐specific primers targeting the intergenic spacer (IGS) region and characterize intraspecific variation and homologous recombination events for isolates obtained from diseased garlic bulbs in Spain. Sixty‐nine isolates were morphologically identified as F. proliferatum, and their identity was confirmed by sequencing of the translation elongation factor; however, specific IGS primers did not result in an amplification product for nine of these isolates. Further analysis showed that this was due to polymorphism in the IGS region and six isolates were classified as IGS type I, while the remaining isolates were type II. Sequencing of the complete IGS region revealed numerous sequence polymorphisms amongst F. proliferatum isolates, and regions of recombination. Duplication and deletion events may have occurred via unequal crossing over during mitotic or meiotic recombination. These results suggest that the IGS region may be too variable as a reliable target for F. proliferatum‐specific identification. [ABSTRACT FROM AUTHOR]