Radish (Raphanus sativus) is an economically important crop grown for its edible roots and leaves. It is a self-incompatible, outcrossing species, making the production of homozygous lines and the development of breeding populations difficult. However, this can be overcome with haploids production techniques using isolated microspores, providing the rapid production of homozygous lines for breeding. Thus, it would be useful to identify radishes with a high regeneration rate from microspore culture. In the current study, 96 radish cultivars or germplasms were evaluated for high regeneration rates. Also, a single-marker analysis (SMA) was applied to detect single nucleotide polymorphisms (SNPs) potentially associated with this trait using genotype-by-sequencing (GBS) technology. The regeneration rate from microspore culture of 96 lines showed a wide range, from 0% to 269.5%. From the SMA, 52 markers were detected at a p value of 0.001 and a total of 11 physically nearby genes with high levels of similarity in various species were identified as candidates for high regeneration rates. This result could be used for clarifying the genetic basis underlying these traits and developing molecular markers associated with regeneration rates and would be beneficial for generating homozygous inbred lines.