ContentsThis work examines the effect of the interaction between different concentrations of two cryoprotectants – glycerol (GLY) and dimethylacetamide (DMA) – and two methods of cryopreservation – pellets produced by plunging into liquid nitrogen and gradual in‐straw freezing – on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6%DMA, following the in‐straw and the pellet methods (ii) 11%GLY, following the in‐straw and the pellet methods; and (iii) 8%GLYin the in‐straw method and 3%DMAin the pellet method (i.e. reduced cryoprotectant concentrations). When 6%DMAwas used as the cryoprotectant, no differences were seen between the in‐straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in‐straw method with 11%GLYwere higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in‐straw and pellet methods, respectively). Finally, the use of 8%GLYin the in‐straw method returned higher sperm viability, intact acrosome and motility values than the use of 3%DMAin the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.