Nicotiana tabacum (tobacco, 2n = 4x = 48) is an allotetraploid with 24 S-genome chromosomes (from a diploid related to N. sylvestris) and 24 T-genome chromosomes (from a diploid related to N. tomentosiformis). The BY-2 suspension cell culture, derived from N. tabacum cultivar Bright Yellow 2, has been used extensively for research in molecular and cell biology for almost 40 years; a Web of Knowledge search reveals that it has been used over 150 times since 2008 alone, largely for cell cycle and plant physiology studies. However, we show that this culture is unstable and, as with other long-term cultures, exists as a community of cells with different karyotypes reflected in different chromosome numbers, morphologies and distributions of satellite repeats, At least one rearranged chromosome type was found in all cells investigated in detail. In comparison with N. tabacum, one satellite repeat, NTRS, has become dispersed across several chromosomes and there is complete homogenization of 35S rRNA genes towards T-genome type rDNA units. Karyotype divergence should be considered when using BY-2 cells for plant physiology or cell cycle/development studies in the future. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 459–471.