Purpose of the researchWhile many inferences have been made about the low degree of genetic variation among intra specific populations of Clitoria ternatea L. (CT), pharmacognostic biochemical investigations using tandem mass spectrometry have never been conducted. Methanol leaf extract of Clitoria ternatea L., populations collected from 13 different agro-climatic zones of India was assessed for identification and characterization of targeted nootropic metabolites, using liquid chromatography tandem mass spectrometry product ion scanning (LC-MS2 PIS). Based on the RI values derived from LC-MS2 (PIS) superior population(s), in terms of the presence of the presence of nootropic metabolites, were identified. The intra-specific correlation among different populations, was analyzed through PCA (principal component analysis), scree plot, correlation matrix and hierarchical cluster analysis. Principal resultsThe targeted mass spectrum obtained by tandem mass spectrometric analysis revealed the presence of acetylcholine, choline, caffeic acid, gallic acid, ferulic acid, kaempferol and D-L valine mass fragments in the leaf of CT. These mass groups are belonging to four classes viz., phenols, flavonoids, neurotransmitters and amino acids. Based on the high degree of relative intensities of targeted nootropic metabolites, the Odisha population, collected from Cuttack, outperformed the rest of twelve populations. Correlation matrix data also revealed that populations habituated in similar climatic conditions show significant correlation with each other. Hierarchical cluster analysis classified the 13 populations in two groups. The first group contained 11 populations and the second group is restrained into two populations; one from Cuttack, Odisha and second one from Udaipur, Rajasthan. Major conclusionThis is the first report to identify the superior population of Clitoria ternatea L. across India, through multivariate statistical analysis of LC-MS2(PIS) for gallic acid, ferulic acid, caffeic acid, kaempferol, D-L-valine, acetylcholine, and choline. Even though earlier studies reported an enhancement of acetylcholine content in the central nervous system. The present work may explain that the plant is a producer of acetylcholine and choline itself so it can directly affect the memory and cognition of the brain. Simultaneously, the study confirms the presence of abovementioned seven nootropic metabolites in the leaf of CT. The Odisha population (collected from Cuttack) transcends over all other 12 populations, with highest RI values of seven nootropic metabolites. According to hierarchical cluster analysis, two populations, viz., the one collected from Cuttack, Odisha and the other collected from Udaipur, Rajasthan were distantly clustered from other eleven populations. Hence, both these populations should be given priority for pharmaceutical supply and value chain management systems.