Abstract: The objective of this study was to compare the effect of fungal genomic DNA, which was extracted by five methods including CTAB method, modified CTAB method, SDS method, benzyl chloride method and Chelex-100 method, as PCR templates. Eleven sooty blotch and flyspeck (SBFS) fungal genera that exhibited different growth rates and colony characters were selected as tested fungi, and amplification percentages of PCR reactions of the internal transcribed spacers (ITS) and partial sequences of 28S rRNA gene of ribosomal RNA (rRNA) were used as indexes, and the results were analyzed with SPSS software. The results showed that the CTAB method and the modified CTAB method were suitable for most fungi, respectively with seven and nine tested fungal genera amplification percentage higher than 70% and had no significant differences. The benzyl chloride method and the Chelex-100 method were suitable for partial fungi, respectively with six and eight tested fungal genera amplification percentage higher than 70% and 50%, and had no significant differences. The SDS method was suitable for only a narrow range of fungi, with six tested fungal genera amplification percentage higher than 50% and had no significant differences. There were at least two methods that could perform well to extract DNA from most SBFS fungal genera, but only the benzyl chloride method gave the best DNA extraction result for the genus Scleroramularia. Using modified CTAB method combined with the benzyl chloride method could meet the need of extracting SBFS fungal genomic DNA as PCR templates.