A total of 297 single-varietal olive oil samples collected from four California regions (Central Valley, Wine Country, South Coast, and Desert) were analyzed on the fatty acid (FA) and sterol compositions between 2010 and 2017. Significantly higher amounts of palmitic acid (PAM), palmitoleic acid, and total sterols were detected in varieties including Arbequina, Arbosana, Picual, Leccino, and Koroneiki from the Desert region. Two super-high density varieties Arbequina and Koroneiki from the Desert region had a significantly higher campesterol content (5.3 AE 0.2 and 4.7 AE 0.1%, respectively) compared to the same varieties from the Wine Country and the South Coast, not meeting the limit of the existing California olive oil standards [Correction added on 8 March 2019 after first online publication: the preceding sentence has been updated to reflect the correct campesterol content.]. The impact of severe droughts in California during late 2011 and 2014 on the major FA and sterol compositions of California olive oil was also analyzed. Multivariate chemometrical tools, one-way ANOVA and principle component analysis (PCA), confirmed the significant differences between samples from the Desert region and other growing regions among several varieties and achieved desirable segregation on the PCA biplots for variety-region classification. This study demonstrates a feasible approach of olive oil composition database establishment for new olive-growing regions; the proposed chemometrical method can be used to characterize unknown variety of olive oils produced from different regions in California; and the collected FA and sterols data provide information for revision recommendations to the current olive oil standards.