Prostate cancer (PCa) is a heterogeneous disease both clinically and genetically. According to The Cancer Genome Atlas (TCGA), the speckle‑type POZ protein (<i>SPOP</i>) mutant form is one of the significant core subtypes of PCa. However, the prognostic value of <i>SPOP</i> variations remains unknown. As a critical PCa driver and an SPOP-targeted protein, androgen receptor (AR) also plays a role in PCa initiation and progression. Thus, we aimed to analyze the mutational status of <i>SPOP</i> and <i>AR</i> with their transcriptional levels in a pathological stage 3 (pT3) prostatectomy cohort consisting of 89 Turkish PCa patients. Targeted sequence analysis and RT-qPCR were performed for <i>SPOP</i> and <i>AR</i> in the benign and malign prostate tissue samples. Our results introduced the two novel pathogenic <i>SPOP</i> variations, C203Y and S236R, in the BTB/POZ domain and a novel pathogenic variant in the ligand-binding domain of <i>AR</i>, R789W. Their predicted pathogenicities and effects on protein features were evaluated by web-based <i>in silico</i> analysis. The overall frequency of <i>SPOP</i> and <i>AR</i> variations for pT3 patients in our population was 3.4% (3/89) and 4.5% (4/89), respectively. The mutational results represented a possible subgroup characterized by carrying the novel variants in <i>SPOP</i> and <i>AR</i> in pT3 PCa patients. In addition to the significant clinicopathological parameters, the mutational results provide a better understanding of the molecular structure of pathologically advanced PCa in the <i>SPOP</i> and <i>AR</i> aspects.