Small cell lung cancer (SCLC) shows an excellent sensitivity to chemotherapy, but commonly develops resistance after a few months. An early identification of a genomic marker in drug discovery may help to select patients who would respond to treatment in clinical trials. Herein, we characterized the parental NCI-H69 (sensitive) and NCI-H69AR (anthracycline-resistant) cell lines by G-banding and spectral karyotyping (SKY). In the H69 cell line, SKY allows us to redefine three alterations that are not well characterized by G-banding and to confirm seven. For H69AR, SKY redefined 10 chromosomal alterations and confirmed four observed by G-banding. Fluorescence in situ hybridization confirmed the amplification of the MYCN gene (dmin or hsr) in these two cell lines, although only the H69AR cell line showed MYCN amplification in the form of homogeneously staining regions. It should be noted that a new derivative chromosome appears in the H69AR cell line, a der(16)t(3;16;18;5;18), characterized by SKY as showing 18q amplification. Amplification of genes located in this region may correlate with resistance to anticancer therapies. We suggest that the 18q marker may have a broader application in SCLC. In conclusion, SKY provides a useful complementary technique to routine cytogenetics for the accurate characterization of SCLC cell lines and could provide some relevant information concerning regions involved in chemoresistance.