Heat shock protein 90 (HSP90) is a molecular chaperone that stabilizes critical client proteins in multiple cancers. Gene expression profiling was utilized to characterize HSP90 isoform expression in primary human diffuse large B‐cell lymphomas (DLBCLs). HSP90 α and β isoforms were differentially expressed in subsets of tumours defined by their transcriptional profiles. Thereafter, we assessed the activity of the HSP90 inhibitor, IPI‐504, in an extensive panel of DLBCL cell lines. IPI‐504, which interacts with the conserved ATP‐binding site in both HSP90 isoforms, inhibited proliferation and induced apoptosis in the majority of DLBCL cell lines at low micromolar concentrations. IPI‐504‐sensitive cell lines expressed high levels of the HSP90 client protein, pAKT, and exhibited dose‐dependent decreases in pAKT levels following IPI‐504 treatment and significantly reduced proliferation following AKT RNAi. Furthermore, the combination of low‐dose (<1 μmol/l) IPI‐504 and the AKT/Pi3K pathway inhibitor, LY24009, was synergistic in IPI‐504‐sensitive DLBCL cell lines. Low‐dose IPI‐504 was also synergistic with the chemotherapeutic agent, doxorubicin. The HSP90 inhibitor IPI‐504 warrants further investigation in DLBCL alone and in combination with identified client protein inhibitors and active chemotherapeutic agents.