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A great diversity of small fish species inhabiting shallow aquatic biotopes of the South American Atlantic Forest have been reported in recent years, but records of interstitial catfishes of the genus Listrura for the southern subtropical Brazilian region are still rare. During field inventories in this region, two new species were collected, which are herein described. The new species are distinguished from congeners by a combination of morphological characters, including caudal peduncle morphology, number of vertebrae, relative position of fins, and bone morphology. The findings here described indicate that the scarcity of records of Listrura for the subtropical Atlantic Forest is probably due to the absence of collecting efforts in specialised biotopes, since species of Listrura are leaf litter dwellers. Both new species are probably more closely related to L. depinnai than to other congeners as all three uniquely possess an expanded skin fold supporting the procurrent caudal-fin rays, making the caudal peduncle deeper than the body region anterior to the dorsal and anal fins. However, only one of the new species shares with L. depinnai the presence of a process on the dorsal surface of the autopalatine articular facet for the mesethmoid, a condition not occurring in any other congener. The two new species are easily distinguished from L. depinnai by the absence of a dorsal fin in the latter species. [ABSTRACT FROM AUTHOR]