DNA flow cytometry of the lymph node metastases from 56 patients was used to retrospectively evaluate the prognostic significance of DNA ploidy in patients with stage III melanoma. The findings were correlated with traditional prognostic factors and patient survival. Multivariate regression analysis revealed that, in addition to the number of positive lymph nodes and patient gender, the DNA index was a significant predictor of patient survival (all p < 0.03). Within this cohort of patients, the patients with the poorest prognoses were those with DNA indices greater than 2 (at least tetraploid), more than three positive lymph nodes, and male gender. There was a significant survival difference among the patients having zero, one, or two of these risk factors (p < 0.001). Our results indicate that DNA analysis of melanoma metastases by flow cytometry provides an additional discriminating factor for predicting patient outcome after therapeutic lymph node dissection. This information may be useful in directing patients with stage III melanoma at higher risk for recurrence into clinical trials of more aggressive systemic adjuvant therapy.