The effects of seed priming on germination and endo-β-mannanase activity at inhibitory and non-inhibitory temperatures in thermosensitive ‘Dark Green Boston’ (DGB) and in thermotolerant ‘Everglades’ (EVE) lettuce were investigated. A single-seed endo-β-mannanase assay was used to follow the enzyme activity during priming. Seeds were primed at 15°C in aerated solutions of polyethylene glycol (PEG) with constant light, then redried. Primed and non-primed seeds germinated 100% at 20°C. At 35°C, non-primed and primed EVE seeds germinated 100%, whereas non-primed seeds of DGB germinated only 4%. During priming, endo-β-mannanase activity increased between 24 and 48 h in EVE and between 24 and 72 h in DGB after the beginning of osmotic imbibition. Endo-β-mannanase activity persisted in primed seeds following seed drying, was detected before radicle protrusion and was present in the micropylar region in front of the radicle tip. Higher enzyme activity was observed in primed seeds and EVE compared with non-primed and DGB seeds. The results suggest that priming may overcome the inhibitory effect of high temperature in thermosensitive lettuce seeds due to increased endo-β-mannanase activity, possibly leading to a weakening of endosperm, thus overcoming thermodormancy.