Aroma volatiles in Basmati-370, Ambemohar-157 (non-basmati scented), and IR-64 (non-scented) rice cultivars were qualitatively and quantitatively analyzed at vegetative and maturity stages to study their differential accumulation using headspace solid-phase microextraction, followed by gas chromatography mass spectrometry (HS-SPME-GCMS) with selected ion monitoring (SIM) approach. In addition, expression analysis of major aroma volatile 2-acetyl-1-pyrroline (2AP)-related genes, betaine aldehyde dehydrogenase 2 (badh2) and Delta(1)-pyrolline-5-carboxylic acid synthetase (P5CS), were studied by real-time PCR. Maximum number of volatiles recorded at vegetative (72-58) than at mature stage (54-39). Twenty new compounds (12 in scented and 8 in both) were reported in rice. N-containing aromatic compounds were major distinguishing class separating scented from non-scented. Among quantified 26 volatiles, 14 odor-active compounds distinguished vegetative and mature stage. Limit of detection (LOD) and limit of quantification (LOQ) for 2AP was 0.001 mg/kg of 2AP and 0.01 g of rice, respectively. 2AP accumulation in mature grains was found three times more than in leaves of scented rice. Positive correlation of 2AP with 2-pentylfuran, 6-methyl-5-hepten-2-one, and (E)-2-nonenal suggests their major role as aroma contributors. The badh2 expression was inversely and P5CS expression was positively correlated with 2AP accumulation in scented over non-scented cultivar.