Fragmented wildlife populations are challenged by limited gene flow that can lead to significant inbreeding. The lion (Panthera leo) population in South Africa’s Hluhluwe-iMfolozi Park (HiP) started from a small founder population of one adult male (1958), one adult female, followed by two lionesses and three cubs (two females and one male; 1965; unrelated to the male). A genetic rescue effort was launched after signs of inbreeding were observed in the 1990s. Sixteen lions (13 females and three males) were translocated into the HiP population from Pilanesberg National Park and Madikwe Game Reserve. We assessed the genetic consequences 10 to 15 years post translocation (n = 91), using microsatellite markers. Structure analysis revealed integration of the translocated animals and evidence of a unique post-translocation genetic cluster. Allelic richness increased from 2.26 to 3.88, and heterozygosity from 0.40 to 0.65. However, overall relatedness within (0.19) and among (0.15) existing prides remained higher than in an open system, and both allelic richness and heterozygosity were declining in later post-translocation generations. Thus, although genetic rescue is a valuable tool for the recovery of inbred, isolated populations, genetic augmentation should be performed at regular intervals to ensure continued population viability.