Abstract Purpose: LKB1 loss is common in lung cancer, but no assay exists to efficiently evaluate the presence or absence of LKB1. We validated an IHC assay for LKB1 loss and determined the impact of LKB1 loss in KRAS-mutant non–small cell lung cancer (NSCLC). Experimental Design: We optimized and validated an IHC assay for LKB1 (clone Ley37D/G6) using a panel of lung cancer cell lines and tumors with known LKB1 mutations, including 2 patients with Peutz–Jeghers syndrome (PJS) who developed lung adenocarcinoma. We retrospectively analyzed tumors for LKB1 using IHC from 154 KRAS-mutant NSCLC patients, including 123 smokers and 31 never-smokers, and correlated the findings with patient and tumor characteristics and clinical outcome. Results: LKB1 expression was lost by IHC in 30% of KRAS-mutant NSCLC (smokers 35% vs. never-smokers 13%, P = 0.017). LKB1 loss did not correlate with a specific KRAS mutation but was more frequent in tumors with KRAS transversion mutations (P = 0.029). KRAS-mutant NSCLC patients with concurrent LKB1 loss had a higher number of metastatic sites at the time of diagnosis (median 2.5 vs. 2, P = 0.01), higher incidence of extrathoracic metastases (P = 0.01), and developed brain metastasis more frequently (48% vs. 25%, P = 0.02). There was a nonsignificant trend to worse survival in stage IV KRAS-mutant NSCLC patients with LKB1 loss. Conclusions: LKB1 IHC is a reliable and efficient assay to evaluate for loss of LKB1 in clinical samples of NSCLC. LKB1 loss is more common in smokers, and is associated with a more aggressive clinical phenotype in KRAS-mutant NSCLC patients, accordingly to preclinical models. Clin Cancer Res; 21(12); 2851–60. ©2015 AACR.