We report on the occurrence of South American suckermouth armored catfishes (Loricariidae) in the Suwannee River basin, southeastern USA. Over the past few years (2009-2012), loricariid catfishes have been observed at various sites in the Santa Fe River drainage, a major tributary of the Suwannee in the state of Florida. Similar to other introduced populations of Pterygoplichthys, there is high likelihood of hybridization. To date, we have captured nine specimens (270-585 mm, standard length) in the Santa Fe River drainage. One specimen taken from Poe Spring best agrees with Pterygoplichthys gibbiceps (Kner, 1854) or may be a hybrid with either P. pardalis or P. disjunctivus. The other specimens were taken from several sites in the drainage and include seven that best agree with Pterygoplichthys disjunctivus (Weber, 1991); and one a possible P. disjunctivus × P. pardalis hybrid. We observed additional individuals, either these or similar appearing loricariids, in Hornsby and Poe springs and at various sites upstream and downstream of the long (> 4 km) subterranean portion of the Santa Fe River. These specimens represent the first confirmed records of Pterygoplichthys in the Suwannee River basin. The P. gibbiceps specimen represents the first documented record of an adult or near adult of this species in open waters of North America. Pterygoplichthys disjunctivus or its hybrids (perhaps hybrid swarms) are already abundant and widespread in other parts of peninsular Florida, but the Santa Fe River represents a northern extension of the catfish in the state. Pterygoplichthys are still relatively uncommon in the Santa Fe drainage and successful reproduction not yet documented. However, in May 2012 we captured five adult catfish (two mature or maturing males and three gravid females) from a single riverine swallet pool. One male was stationed at a nest burrow (no eggs present). To survive the occasional harsh Florida winters, these South American catfish apparently use artesian springs as thermal refugia. In the Santa Fe River, eradication might be possible during cold periods when catfish congregate in spring habitats. However, should Pterygoplichthys increase in number and disperse more widely, the opportunity to eliminate them from the drainage will pass.