Background The use of fine‐needle aspiration (FNA) as a primary tool in the diagnosis of breast carcinoma provides opportunity for early proliferative characterization of the tumor. This study was undertaken to assess DNA ploidy and S‐phase (SPF) fraction by flow cytometry in fine needle aspirates of patients with breast cancer. Method Fifty patients of breast cancer diagnosed on fine needle aspiration cytology (FNAC) and who subsequently underwent either mastectomy or lumpectomy were included. Material obtained by FNAC was subjected to DNA ploidy and SPF analysis. Immunohistochemical estimation of Ki‐67 was done on histopathology sections. The proliferation markers (SPF and Ki‐67) were compared with each other and with the histopathologic parameters. Results On DNA flow cytometry, 27 (54%) cases were aneuploid and 23 (46%) cases were diploid. The median SPF was 12.43% and 4.03% in aneuploid and diploid tumors respectively. Median Ki‐67 among aneuploid tumors was 28.6% compared to 8.7% among diploid tumors. Aneuploid tumors were significantly associated with higher values of SPF and Ki‐67, with Kappa 0.437 and agreement of 72%. Diploid tumors showed lower values of SPF and Ki‐67, with Kappa 0.455 and agreement of 72.7%. Correlation among SPF and Ki‐67 was highly significant with Kappa value 0.446, P value of .002 and agreement of 72.3%. Conclusion DNA ploidy and proliferative activity by flow cytometric SPF estimation on fine needle aspirates from breast cancer can provide valuable prognostic and predictive information at the time of diagnosis in patients with breast cancer. This might help in selection of appropriate treatment modality.