Extramammary Paget’s disease (EMPD) is one of the most intractable malignant tumors of the skin with a high local recurrence rate. Although a discourse called subclinical extension of tumor cells has been advocated and widely accepted, the prevalence of subclinical extension, and the localization, histology, and the topographic distribution of these cells have remained unknown. Since the conventional histological examination is achieved with thin slices, it provides limited information as for the three-dimensional (3D) distribution of tumor cells. Moreover, transformation and shrinkage of tissues hinder a direct comparison between microscopic and macroscopic findings. Two-photon microscopy (TPM) is a laser-scanning imaging technique that enables 3D imaging inside biological tissues at a subcellular resolution. Moreover, recent progress of tissue-clearing significantly increased the observational depth of TPM, and also improved the efficiency of antibody penetration into tissues for whole-mount immunostaining. Within this report, we succeeded in the 3D visualization of tumor cells of EMPD by TPM in combination with a tissue-clearing method and whole-mount immunostaining by anti-cytokeratin 7 antibody. Histological borders were compared with clinical borders defined on dermoscopic images in 14 samples from 11 patients with primary anogenital EMPD. In the 12 samples, histological borders were clearly delineated and strictly corresponded to the clinical borders. However, in 2 samples, tumor cells extended beyond the clinical borders, which fits the notion of subclinical extension. Although the 2D-histological analysis showed a sparse and isolated distribution of tumor cells in the lesion of subclinical extension, 3D visualization of the lesions by TPM revealed a contiguous distribution of tumor cells and clear polygonal borders like other cases with clear clinical borders. Taken together, our method not only verified the subclinical extension in EMPD but also revealed the lining pattern of tumor cells at the edge of subclinical extension.