Abstract. Khaeruddin, Wahjuningsih S, Ciptadi G, Yusuf M, Hermawansyah, Sahiruddin. 2022. Cryopreservation of Gaga’ chicken semen from South Sulawesi, Indonesia with the addition of L-carnitine, hyaluronic acid, sucrose and their combination in diluent. Biodiversitas 23: 3297-3302. Gaga' chicken (Gallus gallus domesticus) is Indonesian germplasm that needs to be preserved with cryopreservation technology. Therefore, this study aims to determine the effect of adding L-Carnitine, hyaluronic acid, and sucrose as well as their combination in a diluent on the quality of Gaga' chicken spermatozoa after freeze-thawing. A completely randomized design was used along with 6 treatments, namely P0, P1, P2, P3, P4, and P5, containing Ringer acetate-egg yolk (RAEY) diluent (P0), RAEY + 0.063 mM hyaluronic acid (P1), RAEY + 1 mM L-carnitine (P2), RAEY + 1 mM sucrose (P3), RAEY + 0.063 mM hyaluronic acid + 1 mM L-carnitine (P4), and RAEY + 0.063 mM hyaluronic acid + 1 mM L-carnitine + 1mM sucrose (P5). Liquid semen was packed in a 0.25 mL straw, equilibrated at 5oC for 2 hours, and placed 3 cm above the surface of liquid nitrogen. It was then immersed in nitrogen for 24 hours and thawed at 60°C for 5 seconds. The results showed that there was a significant difference (P<0.05) in the motility, viability, and kinematic variables of spermatozoa after freeze-thawing. P1, P2, P3, and P4 increased progressive motility and curvilinear velocity, while viability increased with P1, P2, and P4 treatments. Furthermore, the addition of Hyaluronic acid, L-carnitine, or their combination in the RAEY diluent can improve the sperm quality of Gaga' chickens.