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DNA methylation with 5-methylcytosine (5mC) has been reported in the genome of several eukaryotes, with marked differences between vertebrates and invertebrates. DNA methylation is poorly understood as its role in evolution in insects. Drosophila gouveai (cluster Drosophila buzzatii) presents larvae that develop obligatorily in necrotic tissues of cacti in nature, with the distribution of populations in South America, and plasticity of phenotypes in insect-plant interaction. We characterize organisms at developmental stages and analyze variations at multiple methylation-sensitive loci in pupae, and adult flies using methylation sensitive amplification polymorphism. We obtained 326 loci with CCGG targets in the genome of D. gouveai. Genomic regions with molecular lengths from 100 to 700 pb were most informative about methylation states. Multiple loci show differences in methylation-sensitive sites (MSL) concerning developmental stages, such as in pupae (MSL = 40), female reproductive tissue (MSL = 76), and male reproductive tissues (MSL = 58). Our results are the first evidence of genome-wide methylation in D. gouveai organisms., (© 2023 Wiley Periodicals LLC.)