Key message: Loss of viability of recalcitrant seeds upon dehydration is associated with changes in expression levels of genes associated with seed development, the antioxidant defense system and stress responses. Abstract: Araucaria angustifolia is a Brazilian conifer species that produces desiccation-sensitive seeds. Conservation of recalcitrant seeds by conventional seed banking has severe limitations as the underlying mechanisms of quick viability loss upon drying remain unclear. The aim of this study was to shed light on these underlying mechanisms in A. angustifolia by analyzing the transcript levels of genes related to seed development, antioxidant systems and stress responses in seeds subjected to dehydration. To be able to perform these expression analyses, an additional aim was to identify candidate reference genes suitable for gene expression analyses in A. angustifolia. Using GeNorm software we analyzed 10 selected candidate reference genes for RT-qPCR analysis of gene expression in embryonic axis and cotyledon tissues of dehydrated seeds. The most stable candidate reference genes for normalization of gene expression data in heterogeneous samples (inter-tissues) were ACT, GAPDH and SAR1. Slight drying (35% water content) had a positive effect on seed viability since radicle protrusion values (84%) did not differ significantly from undried seeds (94%). Seed viability, however, decreased dramatically when seeds were dried down to a water content of 28%. Transcript levels of ABI3, LEC1, SMP, APX, MIPS and XERO1 decreased in embryonic axis and cotyledons during dehydration, whereas transcript levels of CAT and NAC increased. Our study provides information on reference genes that are adequate for gene expression studies in A. angustifolia seeds and shows that loss of seed viability during dehydration appears to be associated with a decrease in transcript levels of genes related to desiccation tolerance.