The DT40 transformed chicken B-cell line is an important and widely used vertebrate cell line. Knowledge of the 'wild-type' DT40 karyotype is a significant consideration for most research applications and for extrapolation of results to normal cells. Here we present data indicating that (1) modal karyotype differences exist between DT40 cultures from different sources, (2) DT40 cultures are cytogenetically mosaic, and (3) the mosaic features can change over time with media conditions influencing the degree and type of non-modal karyotypes that emerge. In addition to the previously reported trisomy for chromosome 2, a monosomy for chromosome 4 and a variant 4p- are described. The macrochromosomal variation and mosaic nature of the DT40 system should alert researchers using this important cell line to consider whether such variation will impact experimental results and interpretation. The results are discussed in regard to current knowledge of cytogenetic anomalies associated with chicken cancers.