Background Chronic gastrointestinal (GI) morbidity occurs in ≥50% of patients after external beam radiotherapy (EBRT) for carcinoma of prostate (CaP). This prospective, longitudinal study examines which baseline measurements of: 1) homocysteine and micronutrients in plasma; 2) chromosome damage/misrepair biomarkers; and 3) anal and rectal dose volume metrics predict GI morbidity after EBRT. Patients and methods In total, 106 patients with CaP had evaluations of GI symptoms (modified LENT-SOMA questionnaires) before EBRT and at one month, one, two and three years after its completion. Other variables measured before EBRT were: 1) plasma concentrations of homocysteine and micronutrients including caroteinoids and selenium; 2) chromosome damage/DNA misrepair (micronuclei/nucleoplasmic bridge) indices; and 3) mean anal and rectal wall doses and volumes of anal and rectal walls receiving ≥40 Gy and ≥60 Gy. Univariate and multivariate analyzes examined the relationships among: 1) plasma levels of homocysteine and micronutrients; 2) indices of chromosome damage/DNA misrepair; and 3) mean anal and rectal wall doses and volumes of anal and rectal walls receiving ≥40 Gy and ≥60 Gy and total GI symptom scores from one month to three years after EBRT. Results Increased frequency and urgency of defecation, rectal mucous discharge and bleeding after EBRT resulted in sustained rises in total GI symptom scores above baseline at three years. On univariate analysis, total GI symptom scores were significantly associated with: 1) plasma selenium and α tocopherol; 2) micronuclei indices of DNA damage; 3) mean anal and rectal wall doses; and 4) volumes of anal and rectal wall receiving ≥40 Gy and ≥60 Gy (p = 0.08-<0.001). On multivariate analysis, only volume of anal wall receiving ≥40 Gy was significant for increased GI symptoms after EBRT (p < 0.001). Conclusion The volume of anal wall receiving ≥40 Gy predicts chronic GI morbidity after EBRT for CaP.