Abstract Purpose The progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) in humans is highly variable. To better understand the relationship between them, we performed a multi-omic characterization of co-occurring DCIS and IBC lesions in a cohort of individuals. Methods Formalin-fixed paraffin-embedded tissue samples from 50 patients with co-occurring DCIS and IBC lesions were subjected to DNA-seq and whole transcriptome RNA-seq. Paired DCIS and IBC multi-omics profiles were then interrogated for DNA mutations, gene expression profiles and pathway analysis. Results Most small variants and copy number variations were shared between co-occurring DCIS and IBC lesions, with IBC exhibiting on average a higher degree of additional mutations. However, 36% of co-occurring lesions shared no common mutations and 49% shared no common copy number variations. The most frequent genomic variants in both DCIS and IBC were PIK3CA, TP53, KMT2C, MAP3K1, GATA3 and SF3B1, with KMT2C being more frequent in DCIS and TP53 and MAP3K1 more frequent in IBC, though the numbers are too small for definitive conclusions. The most frequent copy number variations were seen in MCL1, CKSB1 and ERBB2. ERBB2 changes were not seen in IBC unless present in the corresponding DCIS. Transcriptional profiles were highly distinct between DCIS and IBC, with DCIS exhibiting upregulation of immune-related signatures, while IBC showed significant overexpression in genes and pathways associated with cell division and proliferation. Interestingly, DCIS and IBC exhibited significant differential expression of different components of extracellular matrix (ECM) formation and regulation, with DCIS showing overexpression of ECM-membrane interaction components while IBC showed upregulation of genes associated with fibronectin and invadopodia. Conclusion While most co-occurring DCIS and IBC were mutationally similar and suggestive of a common clonal progenitor, transcriptionally the lesions are highly distinct, with IBC expressing key pathways that facilitate invasion and proliferation. These results are suggestive of additional levels of regulation, epigenetic or other, that facilitate the acquisition of invasive properties during tumor evolution.