The HeLa cell line is one of the most popular cell lines in biomedical research, despite its well-known chromosomal instability. We compared the genomic and transcriptomic profiles of 4 different HeLa batches and showed that the gain and loss of genomic material varies widely between batches, drastically affecting basal gene expression. Moreover, different pathways were activated in response to a hypoxic stimulus. Our study emphasizes the large genomic and transcriptomic variability among different batches, to the point that the same experiment performed with different batches can lead to distinct conclusions and irreproducible results. The HeLa cell line is thought to be a unique cell line but it is clear that substantial differences between the primary tumour and the human genome exist and that an indeterminate number of HeLa cell lines may exist, each with a unique genomic profile.