Aim:Reference genes are used to normalize target genes in high or low throughput gene expression experiments. It is key to identify a set of stable genes as a reference gene set in sacral and clival originated chordoma cell lines, for cross-comparisons across subtypes. This would allow aggregating datasets from different subtypes in this (and similar) rare cancers. Within this study, we present the best housekeeping genes including clival/sacral based chordoma and nucleus pulposus cells.Material and Methods:To identify the stable genes in chordoma cell lines from sacral and clival subtypes, we investigated 13 candidate reference genes in public chordoma array transcriptome datasets and validated these genes by using RT-PCR and evaluate the stability by NormFinder, geNorm, Bestkeeper. Results:YWHAZ, TBP and PGK1 genes were identified as the most stable reference genes by confirming with three different approaches. On the other hand, KRT8, KRT19 and GAPDH genes are less stable and not appropriate for use in chordoma research. Conclusion:For normalization of RT-PCR experiments in gene profiling of chordoma, we recommend the use of the stable genes YWHAZ, TBP, and PGK1 genes.