In the present study, we investigated the genetic variability among 49 new isolates of trypanosomatids from phytophagous Hemiptera by means of morphological characters, growth features, and biochemical (enzymes of ornithine–arginine cycle) and molecular markers (based on spliced-leader, and ribosomal genes). From 402 phytophagous insects dissected and examined for the presence of trypanosomatids, 228 species belonging to Pyrrhocoridae, Coreidae, Lygaeidae, and Pentatomidae families harbored trypanosomatids in their salivary glands, or digestive tubes. Among these insects, 211 carried promastigotes and only 17 had choanomastigote forms. The results show a strong association among morphology, growth features, and biochemical and molecular markers and reveal the genetic diversity of the isolates, which were assigned to Crithidia, Phytomonas, and Leptomonas; we found genetic polymorphism within all these genera, thus indicating high genetic variability among trypanosomatids from phytophagous insects.