Abstract: Since genomic DNA of high quality is a crucial prerequisite for molecular ecology studies, we specially detected efficient protocols to isolate highquality genomic DNA from ethanolpreserved Pseudaletia separata adults of several natural populations trapped by light traps in fields of different areas. The results of comparative experiment of 4 DNA isolation protocols in 2 mL microcentrifuge tubes showed that only 7.69%-40% of DNA products were qualified in those isolated with conventional phenol extraction protocols. But the qualified rate of DNA products extracted with CTAB+phenol extraction protocols increased to 68.42%-95.28%. Moreover, 5.59-10.04 mg/g DNA was recovered with CTAB+phenol method, much more than 2.83-5.78 mg/g with conventional phenol extraction protocol (significantly different or not from statistical tests in different populations). An additional investigation revealed that abdominal issue was more suitable for DNA extraction than thoracic issue. Correlation analysis of the data set including 99 qualified DNA samples from a natural population indicated that there was a weakly positive correlation between tissue amount (mg) and DNA total yield (μg), a moderately negative correlation between tissue dose (mg) and average DNA yield (mg/g). In conclusion, CTAB+phenol extraction protocol is an efficient method for extracting highquality and highquantity genomic DNA products from 10-20 mg abdominal tissue in a 2 mL microcentrifuge tube, which has been proved suitable for microsatellite identification and genotyping.