The present invention belongs to the technical field of micro-organism genotype identification, particularly relates to a molecular biological identification method of prototheca zopfii and an application thereof, aims at utilizing the molecular biological method to conduct identification and genotyping of the prototheca zopfii, further investigates infection statuses of the pathogen in our region, and carries out researches on prevention and control measures. Main points of a technical scheme for the genotype identification comprise: designing primers according to a target cob gene sequence,wherein the positive and reverse primer sequences of the cob gene sequence of type 1 and 2 type of prototheca zopfii gene; extracting DNA templates to be tested; and using the designed primers and theobtained DNA templates to conduct a PCR reaction and judging the genotypes according to electrophoresis results.