Objective To establish a rapid method for the clinical detection and identification of fungi in clinical urine samples.Methods DNA was extracted from clinically collected urine sample,and the fungal ribosomal internal transcribed spacer was amplified by polymerase chain reaction (PCR) and followed by pyrosequencing.The fungal species were identified by sequence alignment.Results The identification results were compared between PCR-pyrosequencing and conventional culture method.Among the 1320 urine samples,180 were detected positive by conventional method with the positive rate of 13.6％,while 192 were positive by the pyrosequencing based method with the positive rate of 14.5％.The overall coincidence rate of the two methods was 99.09 ％,with the positive coincidence rate of 100 ％ and the negative coincidence rate of 98.95 ％.The Kappa value was 0.963,suggesting a good consistency.The results of 13 standard strains were consistent with the actual results.Conclusions A rapid culturefree method for the detection of fungi in urine sample has been successfully established.This method is based on PCR-pyrosequencing technology with highly accuracy,sensitivity and reproducibility.It is highly automated,cost effective and with high throughput (96 samples per run).The fungal pathogen in urine is identified by single step test within 3 hours without conventional culture.Thus,it is applicable in the clinical laboratory.