Cryopreservation success is usually analysed in terms of cell survival, although there are other potential effects that do not necessarily result in cell death. These include DNA damage, which could result in altered gene expression. Real-time reverse transcriptase PCR allows quantitative analysis of gene expression but usually requires analysis of a 'housekeeping' gene as an internal reference. As the stability of housekeeping genes varies significantly among different groups of samples, it is recommended that those chosen are validated for each different type of sample group. This study aimed to validate housekeeping genes for use in cryopreservation studies of zebrafish embryos. Seven potential housekeeping genes were analysed across fresh and chilled intact embryos and across fresh and frozen isolated blastomeres using the GeNorm and NormFinder software packages. Results suggest that combined use of beta-actin and EF1alpha as housekeeping genes would be suitable for cryopreservation studies on zebrafish embryos and blastomeres.