Double-digested Restriction Site Associated DNA Sequencing (ddRAD) through next-generation sequencing (NGS) generates large numbers of loci for characterizing genome-wide variation among multiple samples using next-generation sequencing. Different combinations of restriction endonucleases (REs) may produce varying size distributions of digested fragments, which affect the number of genotyped loci. Understanding digestion profiles across different species will help in selecting REs for digestion in a particular organism. In this study, we use of genome sequences to compare the in silico digestion profile of 26 combinations of REs in 131 insect species with two simulation programs. The number of digested fragments in the 300–450 bp range increases linearly with the size of the genome. Different species and insect orders showed similar profiles when digested by different combinations of REs in silico, indicating the conservation of digestion by double enzymes in insect genomes. Combinations with NlaIII or TaqαI usually produced higher number of fragments in the range 300–450 bp, while combinations with EcoRI or MluCI produced fewer fragments. The proportion of fragments with the same overhangs at the two ends of digested DNA was higher than those with different overhangs. The two four-base enzyme pairs produced more fragments in the 300–450 bp range than pairs of four-base + six-base enzymes. Experimental digestion of three species from Hymenoptera, Lepidoptera and Thysanoptera showed profiles congruent with in silico expectations. Our results shed light on understanding the digestion profiles of insect genomes and provide guidance on selecting REs for ddRAD projects.