Lithobates catesbeianus is an invasive anuran introduced in Brazil that can affect population and community dynamics of native frogs. However, the mechanisms that allow the establishment of this species in non-native areas are not known. Considering that reproduction is a key feature in the dynamics and establishment of an invasive species in a new environment, this study investigated the reproductive biology of L. catesbeianus in a subtropical area of southern Brazil. One-hundred and four females and 79 males were collected during monthly sampling from June 2008 to May 2009 in the State of Paraná. In the laboratory, sex determination and macroscopic determination of the gonad development stage of each specimen were carried out. Subsequently, the gonads were removed and mass was determined to obtain an individual gonadosomatic index (GSI) for each specimen. We also processed a sub-sample of gonadal tissue for histological analysis. Five stages of ovarian development and four stages of testicular development were determined by microscopic analysis. The maturation curve, using the mean monthly variation of the GSI for females and males, showed that reproductive activity was more intense during spring and summer. The maturation curve of females revealed two reproductive peaks during the study period; one from August to November and another in February. There was little variation in the mean monthly GSI values obtained for males, but with greater values from August to February. The snout–vent length (SVL) of the first maturation (shortest SVL at which 50% of individuals are reproductively active [Vazzoler, 1996]) was estimated for females as 84.5 mm. The SVL of the smallest reproducing male was 76.0 mm.