The aim of this study was to investigate the effects of different numbers of freeze–thaw cycles (0, 1, 2 and 3) on barramundi (Lates calcarifer) fillets. Quality and shelf life of fillets were evaluated using the microbiological and physiochemical analyses. The fillets were individually frozen on a polystyrene dish in an air blast freezer at −80C for 8 h, stored in a freezer at −20C for 20 days and then analysis was carried out on the 21st day from the beginning of frozen storage. The results obtained were compared with those of fresh fillets. This study revealed that the pH, total volatile base nitrogen, total viable counts, and L* and b* values of freeze-thawed fillets increased significantly (P < 0.05) with number of freezing cycles whereas ash, protein content and rheological parameters of fillets decreased significantly (P < 0.05) after three freeze–thaw cycles compared with fresh fillets. Practical Applications The purpose of the study was to assess the effect of repeated freeze thawing on barramundi fillets in order to prevent frozen-thawed related yield losses and deterioration in shelf life. This is essential to improve fillet utilization for better economic returns. The expansion of barramundi markets is presently limited because of quality loss during the freezing process. Surprisingly, little attention has been paid to the freezing techniques of this economically profitable species. However, if the barramundi freezing process could be refined, then quality and shelf life could be significantly extended. The present study demonstrates that it is important to prevent temperature variations or abuse during freezing and transport to avoid the detrimental effect of freezing and thawing so as to extend the quality and shelf life of barramundi fillets.