Second opinion of histological specimens is an important part of the daily routine in anatomic pathology practices. Today, extramural second opinion can be easily obtained by sending still images via an electronic network. The aim of this study was to examine the diagnostic accuracy of second opinion diagnosis based on still images selected from glass slides of 90 archived cases originally referred for extramural second opinion. Two pathologists together diagnosed first the still images (phase 1) and then the glass slides (phase 2). Phase 1 and phase 2 diagnoses were compared with the original second opinion diagnoses (OSODs). The pathologists achieved the same diagnostic results in phase 1 and in phase 2 measured against the OSOD, 67.8% (n = 61) and 68.9% (n = 62) complete agreement, respectively. In 29 cases in phase 1, the diagnoses were discordant with the OSOD. Three cases had incorrect benign diagnoses and 8 cases had incorrect malignant diagnoses. There were 8 false-negative diagnoses regarding malignancy, 6 false-positive diagnoses regarding malignancy, and 4 other discordant diagnoses. Eleven of the 29 discordant diagnoses could have had clinical implications. In interpreting these results, it is important to acknowledge the observer variability in diagnostic histopathology in general. In conclusion, the results support the concept of using still images to obtain second opinion diagnosis.