Abstract: Background and Objective: Nitrotoxins or nitroglycosides are aliphatic nitro compounds, which were detected in some legumes (Papilionoideae). They are important due to mammalian toxicities, attraction of pollinators or seed disperses and repulsion or inhibition of herbivores and microorganisms. Legumes nitrotoxins studies have importance from the phytochemistry, chemotaxonomy, domestic nutrition, ecological adaptations and chemodiversity aspects. In this study nitrotoxins of Acacia farnesiana (L.) Willd., Bauhinia purpurea L., Cassia aphylla Cav. and Prosopis stephaniana Kunth species were reported for the first time that is important for their chemotaxonomy, toxicity and pollination. Materials and Methods: Thirty populations of 13 legume species were collected and identified from various parts of Iran. Their dried leaves analyzed for presence of aliphatic nitro compounds. The qualitative test and quantitative determination for aliphatic nitrotoxins were done using the developed Cooke and modified Williams-Parker methods. Recording the absorption spectrum between 400 and 800 nm was done using Cecill 4400 UV-visible double beam scanning spectrophotometer. Results: Nitrotoxins were detected in 5 species (Acacia farnesiana, Bauhinia purpurea, Cassia aphylla, Prosopis stephaniana and Robinia pseudoacacia) at concentrations ranging from 9-25 NO2 mg g–1 plant. Other examined plant species lacked any nitrotoxins. Conclusion: Nitro compounds studies can show plant chemodiversity throughout the Papilionoideae as chemotaxonomic, toxicity and pollination character.