Background Artificial induction of mutagenesis is effective for genetic resource innovation and breeding. However, the traditional mutation methods for fish breeding are not convenient or safe for daily use. Hence, development of a simple, safe and effective mutagenesis method with a high mutation rate and applicability to multiple fish species, is needed. Results We reported the first successful mutagenesis in a marine aquaculture fish species, Japanese flounder, Paralichthys olivaceus , using a novel atmosphere and room temperature plasma (ARTP) mutagenesis tool. ARTP treatment time was optimized for the fertilized eggs and sperm, respectively. Eggs fertilized for 60 min were treated by ARTP with a radio-frequency power input of 120 W, and the ARTP treatment time was 25 min. Under an ARTP radio-frequency power input of 200 W, the optimal treatment time for sperm diluted with Ringer’s solution by 1:40 v /v was 10 min. The ARTP-treated group presented differences in morphological traits such as body height, total length among individuals at day 90 after hatching. Whole-genome sequencing was used to reveal the mutation features of ARTP-treated individuals collected at day 120 after hatching. In total, 69.25Gb clean data were obtained from three controls and eight randomly selected ARTP-treated individuals, revealing 240,722 to 322,978 SNPs and 82,149 to 86,798 InDels located in 17,394~18,457 and 12,907~13,333 genes, respectively. The average mutation rate reached 0.064% at the genome level. Gene ontology clustering indicated that genes associated with cell components, binding function, catalytic activity, cellular process, metabolic process and biological regulation processes had higher mutation rates. Conclusions ARTP mutagenesis is a useful method for breeding of fish species to accelerate the selection of economically important traits that would benefit the aquaculture industry, given the variety of mutations detected.