Amygdalin is a cynaogenic diglucoside responsible for the bitterness of almonds. Almonds display three flavor phenotypes, nonbitter, semibitter, and bitter. Herein, the amygdalin content of 20 varieties of nonbitter, semibitter, and bitter almonds from four primary growing regions of California was determined using solid-phase extraction and ultrahigh-pressure liquid chromatography electrospray triple-quadrupole mass spectrometry (UHPLC-(ESI)QqQ MS/MS). The detection limit for this method is ≤ 0.1 ng/mL (3 times the signal-to-noise ratio) and the LOQ is 0.33 ng/mL (10 times the signal-to-noise ratio), allowing for the reliable quantitation of trace levels of amygdalin in nonbitter almonds (0.13 mg/kg almond). Results indicate that amygdalin concentrations for the three flavor phenotypes were significantly different (p < 0.001). The mean concentrations of amygdalin in nonbitter, semibitter, and bitter almonds are 63.13 ± 57.54, 992.24 ± 513.04, and 40060.34 ± 7855.26 mg/kg, respectively. Levels of amygdalin ranged from 2.16 to 157.44 mg/kg in nonbitter, from 523.50 to 1772.75 mg/kg in semibitter, and from 33006.60 to 53998.30 mg/kg in bitter almonds. These results suggest that phenotype classification may be achieved on the basis of amygdalin levels. Growing region had a statistically significant effect on the amygdalin concentration in commercial varieties (p < 0.05).