Mangaba tree (Hancornia speciosa Gomes) is a tropical fruit species from Brazil and presents socioeconomic potential. The objective of this study was to determine antioxidant activity, rutin content and genetic similarity among in vivo and in vitro matrices and progenies of six accessions from the Mangaba Active Germplasm Bank of the Embrapa Coastal Tablelands, SE, Brazil. Young leaves of adult matrix plants and in vitro callus methanolic extracts obtained from young leaves resulted in differences between the accessions for the rutin content and high antioxidant activity. The Costa Azul accession, from Bahia, Brazil outstood with values above 3,000 g of dry extract/g of DPPH (2.2-diphenyl-1-picrilhydrazyl) in in vitro callus coming from nodal and internodal segments (3,023.73 and 3,136.80 g of dry extract/g of DPPH, respectively). Rutin was not found in extracts resulting from in vitro callus of mangaba tree. The difference among the values obtained for DPPH analysis are superior when compared with in vivo leaf extract, with the in vitro Costa Azul accession (nodal and internodal) outstanding, concluding that in vitro callus induction can interfere with chemical compounds of the plant. The markers detect genetic similarity under in vitro cultivation conditions. The Costa Azul accession group itself in isolation from others and EC 50 concentrations differentiated between leaf and callus are obtained. O EC 50 is superior in extracts deriving from in vitro callus, with the in vitro Costa Azul accession (nodal and internodal).