Supernumerary or B chromosomes are one of the main causes for numerical chromosomal variation in higher eukaryotes. These extragenetic elements have been studied for more than a century, with the goal of trying to understand their origin, and how they survive as a polymorphism in natural populations. Hypsiboas albopunctatus is a nocturnal hylid frog distributed in the central–eastern part of South America. Previously, variation in chromosome numbers was described for a population from Rio Claro, São Paulo, Brazil, in which a single small-sized, metacentric B chromosome was present in ca. 40% of the analyzed individuals (n = 17). We herein describe the presence of B chromosomes in populations of H. albopunctatus from northeastern Argentina (Corrientes and Misiones provinces), with unusual morphological and structural characteristics. The frequency of B chromosomes varied significantly among analyzed populations. We found four diploid numbers (2N = 22, 22 1B, 22 2B, and 22 3B), and in a few individuals mitotic instability occurred. C banding revealed variations in the heterochromatin (DAPI ) pattern between Bs with similar morphology, indicating the existence of two new structural variants of these supernumerary elements in H. albopunctatus (B1 and B2). Nucleolar organizer regions marked positively on the eighth pair, coincident with the location of ribosomal DNA as demonstrated with fluorescent in situ hybridization, but Bs did not mark positive with these two techniques. Also, fluorescent in situ hybridization with telomeric probes showed no differences in location and intensity between Bs and autosomal chromosomes. The present communication is the first case of B chromosome polymorphisms in hylid frogs and the sixth reported in Anura.