Prostate cancer (PC) is the second most commonly diagnosed type of cancer in males with 1,114,072 new cases in 2015. The MTHFR enzyme acts in the folate metabolism, which is essential in methylation and synthesis of nucleic acids. MTHFR C677T alters homocysteine levels and folate assimilation associated with DNA damage. Androgens play essential roles in prostate growth. The SRD5A2 enzyme metabolizes testosterone and the V89L polymorphism reduces in vivo SRD5A2 activity. The androgen receptor gene codes for a three-domain protein that contains two polymorphic trinucleotide repeats (CAG, GGC). Therefore, it is essential to know how PC risk is associated with clinical features and polymorphisms in high altitude Ecuadorian mestizo populations. We analyzed 480 healthy and 326 affected men from our three retrospective case-control studies. We found significant association between MTHFR C/T (odds ratio [OR] = 2.2; P = 0.009), MTHFR C/T+T/T (OR = 2.22; P = 0.009), and PC. The SRD5A2 A49T substitution was associated with higher pTNM stage (OR = 2.88; P = 0.039) and elevated Gleason grade (OR = 3.15; P = 0.004). Additionally, patients with ≤21 CAG repeats have an increased risk of developing PC (OR = 2.99; P < 0.001). In conclusion, genotype polymorphism studies are important to characterize genetic variations in high altitude mestizo populations.