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Finding optimal sample sizes is critical for the accurate estimation of genetic diversity of large invasive populations. Based on previous studies, we hypothesized that a minimal sample size of 3–8 individuals is sufficient to dissect the population architecture of the harlequin lady beetle, <i>Harmonia axyridis</i>, a biological control agent and an invasive alien species. Here, equipped with a type IIB endonuclease restriction site-associated (2b-RAD) DNA sequencing approach, we identified 13,766 and 13,929 single nucleotide polymorphisms (SNPs), respectively, among native and invasive <i>H. axyridis</i> populations. With this information we simulated populations using a randomly selected 3000 SNPs and a subset of individuals. From this simulation we finally determined that six individuals is the minimum sample size required for the accurate estimation of intra- and inter-population genetic diversity within and across <i>H. axyridis</i> populations. Our findings provide an empirical advantage for population genomic studies of <i>H. axyridis</i> in particular and suggest useful tactics for similar studies on multicellular organisms in general.