Hsp70 is biologically relevant for its chaperon functions. The CX− and CX+ sublines, which derive from the parental colon carcinoma CX2 cell line, are accordingly very similar. They have been reported to be specifically different only in Hsp70 membrane expression, which is associated with immunostimulatory effects. CX−/CX+ have been phenotypically characterized by immunofluorescence studies and Raman spectroscopy combined with robust clustering and multivariate analysis. With the latter we address the potential of overall characterization for CX−/CX+ discrimination and gain molecular insights into Hsp70 differential expression. Due to their strong resemblance, CX− and CX+ show similar mean Raman spectra, which look indiscernible at first. Interestingly, their rather protein-dominated Raman spectra reveal, besides changes in protein and amino acids, very specific changes in DNA/RNA nucleotides involving pyrimidine ring Raman hypochromic effects. Therefore, discriminating CX− from CX+ is ultimately achieved based on principal component scores. Because CX−/CX+ are associated with the same lipid marker, changes in proteins support lipid interactions with regulatory proteins. More importantly, changes observed in nucleobases, which are indicative of DNA/RNA–protein binding interactions, suggest transcription deregulations as participating precursor onsets of different transport mechanisms that lead to Hsp70 differential expression and associated phenotypic variation. Besides immunofluorescence, we have used Raman spectroscopy combined with multivariate analysis within an autologous tumor system for label-free nondestructive cell-subline discrimination, and demonstrate, to our knowledge, the first overall phenotypic monitoring with insights into Hsp70 differential expression. This might well prove to be useful for Raman label-free cell-sorting of the CX−/CX+ sublines.