Prostate cancer (PC) is the most common cancer in men with biologically highly heterogeneous clinical outcomes despite early detection. Therefore, the identification of novel molecular markers that are associated with biological aggressiveness is essential for predicting clinical outcomes and deciding the treatment of PC. We examined the expression of cluster of differentiation 44 (CD44) and heat shock protein 70 (HSP70) in PC cells using immunohistochemistry on tissue microarrays and evaluated their clinicopathological significance. A loss of CD44 expression and HSP70 overexpression were observed in 62 (57.9%) and 54 (50.5%) out of 107 cases of PC, respectively. CD44-negative PC showed more vascular invasion, more extra-prostatic extension, more capsular invasion, higher pT stages, higher pathological tumor stages, higher prostate-specific antigen levels (>20 ng/mL), and higher grades groups. Overexpression of HSP70 was significantly associated with PC with capsular invasion, higher pT stages, and higher pathological tumor stages. The loss of CD44 expression is correlated with tumor invasiveness and higher Gleason grades, reflecting the features of aggressive tumors. Consequently, CD44 could be an important biomarker and a potential therapeutic target.