The silique is an important organ in plant reproduction and maintains biodiversity among species; however, little is known about the regulation of radish (Raphanus sativus L.) silique development. In this study, we conducted research on the radish long silique (ls) mutant and wild-type (WT) radish and compared their morphological and molecular markers. The results showed that the mutant obtained by ethyl methane sulfonate treatment had the following stable characteristics: the lengths of mutant and WT siliques were 20.50 and 9.10 cm, respectively; and the ovule number per silique of the mutant and WT was 9.5 and 4.5, respectively. Polymerase chain reaction (PCR) analysis revealed abundant polymorphisms between the ls mutant and WT in HZ001, SRC9-022, and OIL2F11 simple-sequence repeat molecular markers, and the expression of LS1 and LS2 (RsNAC66). Arabidopsis thaliana-transformed plants with RsNAC66 overexpression were obtained by the floral dip method. Quantitative PCR showed that LS2 (RsNAC66) was more highly expressed in transformed lines than in WT, and the expression of LS2 (RsNAC66) in the transformed lines was higher in siliques than leaves. Phenotypic analysis revealed abnormal ovule numbers and shoots, and altered plant height in the transformed plant. Phenotypic and gene expression analyses showed that LS2 (RsNAC66) plays an important role on silique length and the number of seeds per silique. Together, the results showed that the radish ls mutant is specific and stable, and thus constitutes an excellent research resource for improving the seed yield of radishes.