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The purpose of this study was to investigate the feasibility of differentiating malignant prostate from benign prostatic hyperplasia (BPH) and normal prostate tissue by performing frequency domain analysis of photoacoustic images acquired at 2 different wavelengths.We performed multiwavelength photoacoustic imaging on freshly excised human prostate specimens taken from a total of 30 patients undergoing prostatectomy for biopsy-confirmed prostate cancer. Histologic slides marked by a genitourinary pathologist were used as ground truth to define regions of interest (ROIs) in the photoacoustic images. Primarily, 3 different prostate tissue categories, namely malignant, BPH, and normal, were considered, while a fourth category named nonmalignant was formed by combining the ROIs corresponding to BPH and normal tissue together. We extracted 3 spectral parameters, namely slope, midband fit, and intercept, from power spectra of the radiofrequency photoacoustic signals corresponding to the 3 primary tissue categories.We analyzed data from 53 ROIs selected from the photoacoustic images of 30 patients. According to the histopathologic analysis, 19 ROIs were malignant, 8 were BPH, and 26 were normal. All the 3 spectral parameters and C-scan grayscale photoacoustic image pixel values were found to be significantly different (P < .01) between malignant and nonmalignant prostate as well as malignant and normal prostate.Preliminary results of our ex vivo human prostate study suggest that spectral parameters obtained by performing frequency domain analysis of photoacoustic signals can be used to differentiate between malignant and nonmalignant prostate.