Beak and feather disease virus (BFDV), a circovirus, is the etiologic agent of psittacine beak and feather disease (PBFD), a progressive and often fatal disease in Psittaciformes. Even though neotropical psittacine species are more resistant to clinical infection than Old World species, BFDV is recognized as a threat to immunologically naïve wild psittacine flocks and its epidemiologic control is paramount for conservation efforts in Neotropical species. Samples were collected from multiple psittacine species, including Ara species, Amazona species, and the white-crowned parrot (Pionus senilis) from the only rescue center in Guatemala with formal psittacine rehabilitation and reintroduction programs. A total of 117 birds, with 101 adults and 16 juveniles of unknown sex, were tested for BFDV by means of a real-time polymerase chain reaction (PCR) assay. The BFDV prevalence found in this study was 0%, (95% confidence interval, 0%–6.0%). Seven 2–8-year-old scarlet macaws (Ara macao cyanoptera) with positive results from previous surveys by conventional PCR yielded negative results in this study, suggesting complete infection resolution.