Lung cancer evolves in a multistep process, and its early detection portends a better prognosis. Bronchial washings/brushings and fine-needle aspirations are often used as early screening and cytological diagnosis of lung cancer. In some cases, it is difficult to differentiate morphologically malignant from reactive cells. Epidermal growth factor receptor (EGFR) is a transmembrane receptor overexpressed in high percentage lung cancers, and contributes to tumor growth. Assessing EGFR expression levels by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) may provide critical information of tumor marker abnormalities, assist in the cytological diagnosis, and stratify patients for EGFR inhibitor therapy. Fifty patients with bronchial washings/brushings or fine-needle aspiration specimens, and corresponding histologically confirmed lung biopsies, were studied for EGFR expression with FISH and IHC. Copy numbers of the EGFR gene locus were analyzed with those of chromosome 7 by FISH. EGFR and FISH results were compared to our FISH data with combined EGFR, c-myc, 5p15.2, and chromosome 6 probes in selected cases. Cell blocks, if available, and tissue biopsy sections were used for EGFR IHC. The intensity of IHC was scored, and quantified. Only balanced aneuploidy of EGFR was identified by FISH. Gene amplification was not detected. The chromosomal abnormalities of EGFR were often accompanied by other chromosomal aneuploidies demonstrated in c-myc (8q24), 5p15.2 or 6p, indicating a general genomic instability. About half of the specimens with confirmed malignancy showed EGFR balanced aneuploidy by FISH, and gene copy number was not coupled with protein expression in many cases. The benign or reactive cytology specimens confirmed by biopsies had high specificity by FISH (96%) and IHC (88%). FISH and IHC analysis of EGFR, possibly along with other tumor markers, may be a useful ancillary tool to classify difficult cytology cases and inform clinicians arranging targeted chemotherapy.