Species recognition and reproductive isolation are critical for organisms to prevent expensive and unsuccessful matings. This may be particularly important in closely related species that coexist synchronously in the same habitat, and for which reproductive barriers are not entirely effective. Tetragnatha argentinensis Mello-Leitão, 1931 and T. nitens (Audouin, 1826) are two long-jawed orb weaver spiders whose feeding grounds and reproductive phenology overlap extensively. Since general patterns of sexual behavior observed in the field showed no apparent differences between these two species, we proposed to evaluate the occurrence of heterospecific mating, and explored the occurrence of potential reproductive isolation mechanisms between them by analyzing in fine scale the mating behaviors of each species and sex. We observed only one heterospecific mating, and few or no sexual interactions occurred in other crossed trials. We found that both species showed similar general mating patterns, however, there are some subtle differences between them. In T. nitens, males clasped the female's chelicerae with their own, but the opposite occurred in T. argentinensis. Moreover, males of T. nitens produced fewer hematodochal inflations, lower number of flubs, and shorter insertions than males of T. argentinensis. Females of T. argentinensis vibrated their abdomen at a higher rate. Our results indicate that these sympatric Tetragnatha species have successful reproductive isolation that probably takes place through recognition mechanisms occurring prior to mating. We provide an update on the taxonomic status of T. argentinensis and its distribution, and a new junior synonymy.