•Sugar analysis showed 10 more carbohydrates beside glucose, sucrose and fructose.•Total of 49 polyphenolic compounds were identified using LTQ Orbitrap MS.•25 compounds were compared with standards and other confirmed by exact mass search.•Possible markers of botanical and geographical origin of Serbian paprika were found. Spice peppers (Capsicum annuum L.) var. Lemeška and Lakošnička paprika were investigated to evaluate their polyphenolic and carbohydrate profiles and antioxidant activity. A total of forty-nine polyphenolics were identified using ultrahigh-performance liquid chromatography (UHPLC) coupled to LTQ OrbiTrap mass analyzer. Twenty-five of them were quantified using available standards, while the other compounds were confirmed by exact mass search of their deprotonated molecule M−H− and its MS4 fragmentation. Thirteen carbohydrates were quantified using high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). Radical scavenging activity (RSA) ranged from 17.32 to 48.34mmol TE (Trolox equivalent)/kg DW (dry weight) and total phenolics content (TPC) was ranged between 7.03 and 14.92g GAE (gallic acid equivalents)/kg DW. To our best knowledge, five polyphenolic compounds were for the first time tentatively identified in paprika: 5-O-p-coumaroylquinic acid, luteolin 7-O-(2″-O-pentosyl-4″-O-hexosyl)hexoside, quercetin 3-O-(2″-O-hexosyl)rhamnoside, isorhamnetin 3-O-6″-O-(5-hydroxyferuloyl)hexoside-7-O-rhamnoside, and luteolin 7-O-2″-O-(5′″-O-sinapoyl)pentosyl-6″-O-malonylhexoside.