[Objectives]  To investigate the effect of mating frequency and male mating order on the paternity of Spodoptera litura, an important global agriculture and forestry pest. [Methods]  Paternity was determined using five pairs of microsatellite markers. 271 samples were collected, including 21 from female parents, 40 from male parents and 210 from offspring. [Results]  The average number of alleles, expected heterozygosity and polymorphism content information were 4.800, 0.647 and 0.596, respectively. Confidence in estimated paternity was overall > 80%; > 95% for 106 offspring. If a female mated once with two males, 78% of the resultant offspring were the progeny of the last male to mate. If a female mated with a male twice with a male, then with another male once, 50% of the offspring were the progeny of each male. If a female mated with a male once, then with another male twice, 90% of the offspring were the progeny of the last male to mate. If a female mated once with three different males, 5%, 20% and 75% of the offspring were the progeny of the first, second and last males to mate, respectively. [Conclusion]  Both the frequency and order of mating affect paternity in S. litura. Males that mate more than once, and those that mate last, have more progeny than those that only mate once or that aren’t the last to mate.