AbstractSingle nucleotide variants (SNVs), including single nucleotide polymorphisms, are often associated with morphological and/or physiological abnormalities in various organisms. Targeted genomic DNA can be amplified and directly sequenced to detect these mutations, but this method is relatively time consuming and expensive. We recently established the heteroduplex mobility assay to detect genetic mutations as an easy, low‐cost method in genome editing, but detecting such small genetic differences remains difficult. Here, we developed a new, simple method to detect single nucleotide changes in the zebrafish genome by polymerase chain reaction (PCR) and electrophoresis. We first designed a specific single stranded DNA with four tandem guanine nucleotides inserted beside the mutation site, called guanine‐inserted primer (GIP). When reannealing, hybridized complexes of GIP and PCR amplicons with or without 1‐bp‐mutated alleles form different bulge structures, presumably leading to different mobilities on a polyacrylamide gel. This GIP‐interacting mobility assay is easy to use; therefore, it could contribute to the detection of SNVs in any organism.