Due to its potential adverse effects on human health, perfluorooctanoic acid (PFOA), one of the once widely used legacy per- and polyfluoroalkyl substances (PFASs), has been recently replaced by its novel alternatives including hexafluoropropylene-oxide-dimer-acid (GenX) and ammonium 4,8-dioxa-3H-perfluorononanoate (ADONA). These alternative PFASs are detected in water and exposed workers. PFASs can enter organs like thyroids, however, it is yet unknown whether the new alternatives are safer than PFOA. In the current study, we compared the thyroid disrupting effects of PFOA and its alternatives GenX and ADONA in vitro with both rat thyroid cell line FRTL5 and primary normal human thyroid (NHT) cells. Cells were exposed to ascendant doses of PFOA, GenX or ADONA for various incubation time and cell viability was assessed by WST-1 assay and LDH assay. The proliferation rate of survived cells was determined by crystal violet-based cell proliferation assay and MTT assay. The gene expression of thyroid hormone regulation-related genes in thyroid cells after exposure was quantified by RT-PCR and Western blot. Our data showed that both PFOA and GenX reduced thyroid cell viability in both dose and time dependent manner, with GenX being more toxic than PFOA at the same condition. Similarly, the proliferation rate of cells survived exposure to PFOA and GenX was considerably impaired, with GenX showing more profound adverse effect than PFOA. Unlike PFOA and GenX, ADONA showed no apparent adverse effects on the viability and proliferation of both thyroid cell types. Gene expression data revealed that all three PFASs altered gene expression in both thyroid cells and the altered gene expression seemed to be PFAS and cell type dependent. Taken together, our data reveal that the thyroid disrupting effects is increased in the order of GenX > PFOA > ADONA. Our findings will be beneficial for the guidance of the future usage of PFASs and development of better alternatives.