Seed storage under controlled temperature and humidity maintains the viability and the vigor of seeds for a long time while preserving their longevity. Ex situ conservation in a seed storage chamber is perfect for simulating the cool dry conditions that are associated with long-term seed storage. This helps to preserve the availability of seeds for the recovery of degraded areas and to ensure a supply of propagating material for various purposes in silvicultural production, or as raw materials for biotechnological use. The present work evaluated the physical and physiological quality of Enterolobium contortisiliquum (Vell.) Morong seeds from an ex situ collection that was kept in a seed storage chamber at 6 °C ± 3 °C and with 60% ± 5% of RH in waterproof packaging. The water content, the physical quality by X-ray tests, germination, electrical conductivity, and the integrity and quality of RNA that was extracted from the embryonic axes were all determined. It was possible to identify full, damaged, malformed, translucent, and empty seeds. The seeds that had been stored for 1, 5, 9, and 10 years presented 78, 82, 42, and 38% of normal seedlings, respectively, while the lots of 6 and 7 years presented 0 and 6%. The seed storage conditions were the same for all of the lots. A water content higher than 6% influenced the quality of the seeds. The E. contortisiliquum seeds demonstrated high longevity. They could be stored for up to 10 years under controlled conditions.