Most tissue microarray studies have used a single 0.6-mm tissue core per donor tissue. It has been suggested that multiple cores per donor can increase the representativity of tissue microarray studies. To estimate the potential benefit of multiple cores, we analyzed Ki67 and p53 in triplet cores taken from three different areas of 3,261 prostate cancer tissue blocks. Both p53 and Ki67 labeling index were linked to advanced tumor stage (p<0.0001 each), Gleason score (p<0.0001), and early PSA recurrence (p<0.0001) independently of whether the 3 tissue spots were analyzed separately or combined for a consensus result. The rate of positive findings increased with the amount of analyzed tissue. The average Ki67 labeling index was higher in tumors with 3 interpretable spots (5.3±5.6) as compared to two (4.1±4.7) or one interpretable spot (4.1±4.2, p<0.0001). For p53, tumors with three interpretable spots were positive in 3.8% of cases, and tumors with 1 or 2 interpretable spots in 1.9% only (p=0.003). These data demonstrate that using multiple cores in a tissue microarray does not necessarily increase the ability to identify associations of biomarkers with tumor phenotype and prognosis but has always the disadvantage of additional work and tissue requirements. Multiple cores may even lead to statistical problems if unequal amounts of tissue are analyzed per tumor.