Mosquitofish(Gambusia affinis)is a globe invasive species. In order to analyze why they can invade so quickly all over the world, 11 polymorphic microsatellite loci were isolated and employed to examine paternity patterns in 755 samples of 53 families from 10 locations in 3 rivers basins. After sequence genetic analysis of the multiplex SSR PCRs, we got the best fullsib family and analyzed the phylogenetic relationship. We found that there are multiple inseminations in G. affinis. The female in the same womb can reproduce a number of the juveniles, up to 72, with different males, even up to 9. The max fullsib is 41, but the breeder's size isn't the max even not bigger than others. At the same time the max size of the breeder isn't the female which reproduce the largest number of the juveniles. The phylogenetic tree shows that the evolution was far away the sea. The 10 populations can be obviously divided into 3 clusters. The conclusion can be drawn that the key reason of the invasion is G. affinis multiple inseminations. It is suspected that the fertilized frequency isn't proportional to the size of female. Their gene flows rapidly specifically when they were in the migratory distance.