Spectrophotometric intracutaneous analysis (SIAscopy) is an imaging technique developed for diagnostics of pigmented skin lesions. By image analysis, the displayed images indicate the potential distribution and position of melanin, blood, and collagen within the lesion. A topographic comparison was performed between SIAscopic findings and histopathology. In total, 60 patients with suspicious pigmented skin lesions were included. The lesions were SIAscopically imaged and documented before excision and histopathological preparation. Topographical comparisons between SIAscopy findings and histopathology were made. A sensitivity and specificity of 24% and 84%, respectively, were obtained for invasive melanomas. The positive and negative predicted values were 58% and 54%, respectively. The features indicating dermal melanin, blood displacement and collagen holes did only show "no" to "slight" agreement with histopathology, i.e., κ ≤ 0.21. It was concluded that (i) SIAscopy-based diagnosis has low diagnostic accuracy for melanoma, (ii) single SIAscopic features do not provide reliable diagnostic information relating to the lesions internal structure on histopathology examination and (iii) SIAscopy cannot be used as a guide for localizing the maximum tumor thickness when performing the histopathological examination. The importance of validating new optical tools for tumor diagnostics with histopathological findings was demonstrated.