Mycoplasmas are known as commensals and pathogenic bacteria of various raptor species causing clinical or subclinical infections. However, little is known about the prevalence of mycoplasma in captive and wild raptors and its significance to their health. In Israel, the Griffon vulture (Gyps fulvus; hereafter Griffons) is considered critically endangered, and its intensive management program includes population monitoring and restocking (captive-born or imported rehabilitated wild Spanish Griffons). Here we survey the prevalence ofMycoplasmaspecies in both the wild and captive populations. During 2019-2020, we collected 244 tracheal swab samples from 167 unique individuals. We used PCR analysis to identifyMycoplasmaspecies. First, we identified nine spp., including species not yet described in Israel or for Griffon vultures. Second, imported Griffons showed a higher prevalence and a different diversity of species in comparison to the local ones, suggesting that at least oneMycoplasmaspecies (Sp 18b) was introduced into the native population. Third, juvenile Griffons had a higher prevalence, different species composition, and stronger reduction in movement compared to adults, confirming the susceptibility of this group to mycoplasma infections. GPS-tracking of 60 free-ranging individuals showed that even in the absence of apparent clinical signs, Griffons infected with mycoplasma, and especially sub-adults flew less (shorter distances and periods). These findings underscore the importance of considering potential pathogen introductions in population reinforcement and reintroduction initiatives, providing valuable insights for similar conservation programs globally. Further, they demonstrate the potential of long-term tracking for detecting subclinical effects that are unnoticeable in clinical examination.