Thyroid follicular adenomas (FA) and carcinomas (FC) have a high prevalence of aneuploidy. We examined the contribution of mitotic checkpoint dysfunction and mutations of BUB1 or BUBR1, components of the spindle assembly checkpoint pathway, to chromosomal instability in thyroid cancer. The integrity of the mitotic checkpoint was studied in 8 aneuploid thyroid tumour cell lines. All cell lines as well as 9 FA, 9 FC, and 1 aneuploid papillary carcinoma were screened for mutations of BUB1 by SSCP and direct sequencing. Cell lines were also examined for mutations of BUBR1. Neither FRO, NPA nor WRO cells arrested in mitosis after treatment with nocodazole, whereas other aneuploid cell lines paused appropriately following microtubule disruption. One FC had a 2-bp somatic deletion (G2480/A2481) of BUB1 leading to a frameshift, and one FC had a silent polymorphism at nucleotide 1049 (TGT-TGC). There was a silent polymorphism of BUBR1 (G1271A) in one sample. Some, but not all thyroid cancer cell lines with aneuploidy have an abnormal mitotic checkpoint, indicating that chromosomal instability may arise through alternative cell cycle defects. Moreover, mutations of BUB1 or BUBR1 are infrequent in follicular neoplasms, and do not account for aneuploidy in thyroid cancer.