Abstract: In order to rapidly analyze neophytadiene in tobacco leaves, a back blowing column system (5 m DB-5ms pre separation column combined with 10 m DB-5ms analysis column) was developed. Chromatographic conditions (back blowing pressure of 103 kPa and back blowing time of 4.5 min) and pretreatment method (extracting 0.5 g tobacco powder with 10 mL dichloromethane vortexing for 5 min) were established, 114 representative tobacco samples at home and abroad were analyzed. The results showed that: 1) In a range of 10-200 μg/mL, neophytadiene had good linear relationship (r>0.999 9). The limit of quantitation of neophytadiene was 16 μg/g. The spiked recoveries ranged from 98% to 101%, the relative standard deviations (RSD) of inter day and intra day were 1.8% and 1.76% respectively and the long-term (Two-year) RSD was less than 5%. The analysis time of each sample was only 8 min; 2) The contents of neophytadiene in tobacco leaves from five domestic ecological regions and abroad were compared, and no significant differences were found between the southwest plateau ecological region and abroad, but significant differences were observed between the other four domestic ecological regions (Wuling Qinba, Nanling hills, Wuyi hills, Yimeng hills) and abroad; 3) By analyzing the neophytadiene in tobacco leaves from the whole or each individual ecological region, no significant differences were found between the upper and middle leaves, but when compared with the lower leaves, there were significant differences and the content of neophytadiene in the lower leaves was higher. In this study, neophytadiene in tobacco leaves was detected by back blowing gas chromatography and flame photometric detector. The method was simple, rapid, sensitive and reproducible. It can be used for batch quantitative determination of neophytadiene in different parts of tobacco leaves from different production regions.