A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-β. Logically, if the FR positive cell population in a tumor mass is dominated by FR-β positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-β, no study to date has investigated expression of both FR-α and FR-β in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-β (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-β combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-β while pancreatic cancers express primarily FR-β. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-β should be obtained to predict the potential efficacy of a folate-targeted drug.