Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the β‐tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in β‐tubulin isotypes in vitro and has sequenced the β‐tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF‐7 (oestrogen‐receptor positive) and MDA‐MB‐231, (oestrogen‐receptor negative) were made resistant to docetaxel in vitro. Expression of β‐tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT‐PCR and western analysis, respectively. DNA sequencing evaluated the β‐tubulin gene. At the mRNA level, class I, II, III, and IVa β‐tubulin mRNA isotypes were over‐expressed in docetaxel‐resistant MCF‐7 cells when compared with the docetaxel‐sensitive parental cells. However, class VI β‐tubulin mRNA isotype expression was decreased in resistant cells. In MDA‐MB‐231 cells, there was a decrease in expression of the class I and class IVa β‐tubulin mRNA. However, there were increased expressions in class II, IVb, and VI β‐tubulin mRNA isotypes in resistant cells. Western analysis has confirmed corresponding increases in β‐tubulin protein levels in MCF‐7 cells. However, in MDA‐MB‐231 cells, there were decreased protein levels for class II and class III β‐tubulin. This study demonstrates that altered expression of mRNA β‐tubulin isotypes and modulation of β‐tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel.