The heterogeneity among multiple ductal carcinoma in situ (DCIS) lesions within the same patient also diagnosed with invasive ductal carcinoma (IDC) has not been well evaluated, leaving research implications of intra-individual DCIS heterogeneity yet to be explored. In this study formalin-fixed paraffin embedded sections from 36 patients concurrently diagnosed with DCIS and IDC were evaluated by immunohistochemistry. Ten DCIS lesions from each patient were then randomly selected and scored. Our results showed that expression of PR, HER2, Ki-67, and p16 varied significantly within DCIS lesions from a single patient (P<0.05 for PR; P<1×10(-8) for HER2, Ki-67 and p16). In addition, seventy-two percent of the individuals had heterogeneous expression of at least 2/6 markers. Importantly, by evaluating the expression of promising DCIS risk biomarkers (Ki-67, p53 and p16) among different DCIS subgroups classified by comparing DCIS molecular subtypes with those of adjacent normal terminal duct lobular units (TDLU) and IDC, our results suggest the existence of a highly-aggressive DCIS subgroup, which had the same molecular subtype as the adjacent IDC but not the same subtype as the adjacent normal TDLU. By using a systematic approach, our results clearly demonstrate that intra-individual heterogeneity in DCIS is very common in patients concurrently diagnosed with IDC. Our novel findings of a DCIS subpopulation with aggressive characteristics will provide a new paradigm for mechanistic studies of breast tumor progression and also have broad implications for prevention research as heterogeneous pre-invasive lesions are present in many other cancer types.