<b><i>Background:</i></b> Individuals with <i>NR5A1</i> mutations encoding steroidogenic factor-1 (SF1) develop a phenotypically broad range of disorders of sexual development (DSD). Based on a literature review, we noted that hypoplastic seminiferous tubules and the emergence of Leydig cells with vacuolar cytoplasms are seen predominantly in the majority of individuals with <i>NR5A1</i> mutations. <b><i>Aim:</i></b> The aim of this study was to address whether the histopathological characteristics of the testis can be a biomarker for 46,XY individuals with <i>NR5A1</i> mutations. <b><i>Design:</i></b> In order to ascertain whether or not the histological features were the characteristics of <i>NR5A1</i> mutations, we screened the testicular histology of 242 patients with 46,XY DSD and then subsequently assessed <i>NR5A1</i> mutations. <b><i>Result:</i></b> Of 242 patients with 46,XY DSD, 6 patients matched histological testicular features: a reduced number of thin seminiferous tubules and focal aggregations of Leydig cells that contained cytoplasmic lipid droplets. All 6 patients had <i>NR5A1</i> mutations. These histological features were distinct from those of other DSD. Thus, this unique testicular histology is useful for identifying <i>NR5A1</i> mutations in 46,XY patients with DSD before puberty.