Adaptation to cool environments is a common feature in the core group of the grass subfamily Pooideae (Triticeae and Poeae). This suggest an ancient evolutionary origin of low temperature stress tolerance dating back prior to the initiation of taxonomic divergence of core Pooideae species. Viewing the Pooideae evolution in a palaeo-climatic perspective reveals that taxonomic divergence of the core Pooideae group initiated shortly after a global super-cooling period at the Eocene-Oligocene boundary (approximately 33.5-26 Ma). This global climate cooling altered distributions of plants and animals and must have imposed selection pressure for improved low temperature stress responses. Lineage-specific gene family expansions are known to be involved in adaptation to new environmental stresses. In Pooideae, two gene families involved in low temperature stress response, the C-repeat binding factor (CBF) and fructosyl transferase (FT) gene families, has undergone lineage-specific expansions. We investigated the timing of these gene family expansions by molecular dating and found that Pooideae-specific expansion events in CBF and FT gene families took place during Eocene-Oligocene super-cooling period. We hypothesize that the E-O super-cooling exerted selection pressure for improved low temperature stress response and frost tolerance in a core Pooideae ancestor, and that those individuals with multiple copies of CBF and FT genes were favoured.