We investigated whether DNA fragmentation in two cytometric sperm populations (PIdimmer and PIbrighter) with different biological characteristics and clinical relevance is related to clinical and color-Doppler ultrasound (CDUS) parameters of the male genital tract. One hundred and sixty males of infertile couples without genetic abnormalities were evaluated for clinical, scrotal, and transrectal CDUS characteristics, presence of prostatitis-like symptoms (with the National Institutes of Health-Chronic Prostatitis Symptom Index) and sperm DNA fragmentation (sDF) in PIdimmer and PIbrighter populations (using TUNEL/PI method coupled with flow cytometry). Data were adjusted for age (Model 1) along with waistline, testosterone levels, smoking habit, and sexual abstinence (Model 2). According to the statistical Model 2, PIdimmer sDF was associated with testicular abnormalities, including lower clinical and ultrasound volume (r = -0.21 and r = -0.20, respectively; P < 0.05), higher FSH levels (r = 0.34, P < 0.0001) and occurrence of testicular inhomogeneity (P < 0.05) and hypoechogenicity (P < 0.05). PIbrighter sDF was associated with prostate-related symptoms and abnormal signs, including higher NIH-CPSI total and subdomain scores, a higher prevalence of prostatitis-like symptoms and of CDUS alterations such as macro-calcifications, severe echo-texture inhomogeneity, hyperemia (all P < 0.05), and higher arterial peak systolic velocity (r = 0.25, P < 0.05). Our results suggest that DNA fragmentation in PIdimmer sperm, which is related to poor semen quality, mainly originates in the testicles, likely due to apoptosis. Conversely, DNA fragmentation in PIbrighter sperm appears to mainly originate during or after transit through the prostate, increasing with the presence of an inflammatory status of the organ. These results could lead to new perspectives for the identification of therapeutic targets to reduce sDF.