Sex has not received enough attention as an important biological variable in basic research, even though the sex of cells often affects cell proliferation, differentiation, apoptosis, and response to stimulation. Knowing and considering the sex of cells used in basic research is essential as preclinical and clinical studies are planned based on basic research results. Cell lines derived from tumor have been widely used for proof-of-concept experiments. However, cell lines may have limitations in testing the effect of sex on cell level, as chromosomal abnormality is the single most characteristic feature of tumor. To examine the status of sex chromosomes in a cell line, 12 commercially available gastric carcinoma (GC) cell lines were analyzed using several different methods. Loss of Y chromosome (LOY) accompanied with X chromosome duplication was found in three (SNU-484, KATO III, and MKN-1) out of the six male-derived cell lines, while one cell line (SNU-638) showed at least partial deletion in the Y chromosome. Two (SNU-5 and MKN-28) out of six female-derived cell lines showed a loss of one X chromosome, while SNU-620 gained one extra copy of the X chromosome, resulting in an XXX karyotype. We found that simple polymerase chain reaction (PCR)-based sex determination gives a clue for LOY for male-derived cells, but it does not provide detailed information for the gain or loss of the X chromosome. Our results suggest that carefully examining the sex chromosome status of cell lines is necessary before using them to test the effect of sex on cell level.