Cryptolaemus montrouzieri (mealybug destroyer) is a predatory ladybird of Australian origin and has been introduced worldwide to control mealybugs. However, no simple sequence repeat (SSR) marker has been developed for C. montrouzieri, hampering study of its population genetics. We developed and characterized 12 novel SSR markers from expressed sequence tags (ESTs) in C montrouzieri, which proved to be sufficiently polymorphic for population genetic analysis. The results of a population genetic analysis using these markers reveal strong genetic differentiation in ten native and introduced populations, which were not seen in our previous results using mitochondrial markers. Four populations probably underwent a recent genetic bottleneck and one wild population was probably the result of an admixture of two lab-reared populations. Our results from these newly developed EST-SSR markers suggest a strong genetic differentiation among populations of this widely introduced biological control agent. Greater attention should be paid to bottleneck effects, mito-nuclear discordance and admixture for further population genetic analysis, prediction of invasiveness and practical use of the ladybird in biological control. (C) 2017 Elsevier Inc. All rights reserved.