De novo mutations (DNMs) are spontaneous genetic alterations not present in parents but occurring in the germline and transmitted to offspring. These mutations play a vital role in understanding evolutionary processes, genetic diversity, and impacts on species’ fitness and survival. The identification of DNMs is especially crucial in the context of environmental stresses, such as those induced by nuclear accidents like Fukushima. This chapter describes recent advances in high-throughput sequencing technologies that have enhanced our capacity to detect DNMs within a single generation. Due to challenges associated with whole-genome resequencing in nonmodel organisms with high genetic and environmental heterogeneity, we proposed the use of genetic marker-based methods like Restriction site-Associated DNA Sequencing (RADSeq). RADSeq simplifies the detection of DNMs by using restriction enzymes to cut DNA at specific sites, producing manageable fragments for sequencing that represent the entire genome. This method allows for the targeted analysis of genetic variations across large populations and is particularly effective in detecting DNMs from wide range of populations, providing insight into damages and adaptations required for different biological conditions.