Abstract: Maternal haploid (MH) was obtained by the interspecific hybridization method. In this system, Nicotiana tabacum was hybridized as a female with Nicotiana africana due to parthenogenesis. In order to overcome the problems such as low induction rate of maternal haploid and difficult identification of maternal haploid from aneuploid interspecific hybrids in breeding practice,flow cytometry (FCM) was adopted to identify the maternal haploids of hybrid progenies produced by different bagging means at different pollination time. The results showed that maternal haploids were accurately and quickly identified by FCM at seedling stage and eliminated the disturbances from diploids and aneuploid interspecific hybrids. The survival rates of non-haploid seedlings bagged with paper tube and common paper bag were 0.24% and 1.56%,respectively. The identification efficiency of paper tube bagging means for haploid was higher. The suitable time for hybridization was identified as when the difference between sepal length and corolla length(SCD)was 2.5 cm,which was beneficial to the induction of maternal haploids. Therefore,the better way for obtaining maternal haploid was pollination at SCD of 2.5 cm,adopting paper tube bagging means and analyzing progeny ploidy with FCM.