Significance This work demonstrates that HK2-based assay can resolve a novel HK2 ^high /CK ^neg CTC population with consistent genomic CNV but distinct transcriptome signatures compared to the CK ^pos counterpart in NSCLC patients. CK expression levels are found independent of cellular EMT status in these CTCs and may be related to distinct dissemination mechanisms in different types of body fluids. Selective association of CK subtypes in CTCs with patient EGFR mutation types may contribute to suboptimal EGFR inhibitor therapeutic efficacy in EGFR ^L858R mutant tumors, enabling prediction of patients with poor prognosis before therapy. More generally, HK2, as a metabolic function–associated marker, is likely to be useful in identifying CTCs from patients with a wide variety of cancers, independent of epithelial traits.