A beluga (Delphinapterus leucas) sperm preservation method was developed for use in genome banking and AI. In Study 1, glycerol-based cryodiluents (modified BF5F and modified Platz Diluent Variant (PDV)) were unable to maintain adequate progressive motility using straws (fast and slow freezing rate (FR)) or pellets (slow FR). Neither freezing method nor FR affected in vitro sperm characteristics (P > 0.05), but retention of prefreeze progressive motility following thawing was greater (P < 0.05) for BF5F (21%) than PDV (15%). In Study 2, examining the effects of straw freeze-thawing using BF5F with glycerol (1 and 3%, v/v) or trehalose (46 and 91 mM) on sperm characteristics, samples cryopreserved in trehalose exhibited superior (P < 0.05) in vitro parameters compared with their glycerol-treated counterparts. In Study 3, compared with a straw method, directional freezing using 91 mM trehalose enhanced (P < 0.05) sperm characteristics, with samples retaining 38%, 75% and 61% of their prefreeze progressive motility, curvilinear velocity and viability, respectively. A higher (P < 0.05) proportion of motile spermatozoa displayed rapid velocity after directional (21 +/- 1%) compared with straw (12 +/- 3%) freezing. Systematic development of a cryodiluent and the use of directional freezing resulted in beluga spermatozoa exhibiting adequate post-thaw quality for genome banking and use in AI.