The intra-tumour heterogeneity in different locations in the prostate was determined by karyometric image analysis and compared with local tumour progression in a retrospective analysis of 65 patients with localized adenocarcinoma of the prostate. In these 65 radical prostatectomy specimens 290 tumour locations were documented. In each location tumour volume was estimated and Gleason grade determined. Quantitative image analysis of nuclear size, shape and chromatin pattern was performed. Differences in Gleason grade and karyometric feature values were evaluated as measures for intra-tumour heterogeneity. Moreover, the location within the prostate was documented for each tumour area. Gleason grade and karyometric features varied widely in the prostate. Tumour locations in the apex (n = 92) had significantly larger and more irregularly shaped nuclei (P < 0.005) compared with basally located tumours (n = 49). Significant differences in nuclear shape were also found in different locations in the equatorial plane of the organ. Tumour heterogeneity in chromatin pattern features was found to correlate with local extension (seminal vesical invasion, extracapsular tumour growth, positive resection margins) and lymph node metastases. This correlation was even stronger when more pathological features were present. In view of the observed tumour heterogeneity, (karyometric) analysis of multiple tumour areas in the prostate is advisable for optimal evaluation of prostate carcinoma.