Thirty-four species representing all subfamilies and tribes of the Rutaceae Juss. were included in phylogenetic analyses that utilize six molecular data sets from five chloroplast markers, three from the noncoding region (the rps16 gene intron, the trnL–trnF intergenic spacer, and the atpB–rbcL spacer) and two from coding genic regions (rbcL, atpB), with the sixth marker from the Xdh nuclear gene. Based on the large number of nucleotide characters from the chloroplast and nuclear regions as well as the high levels of resolution and support from both parsimony and Bayesian analyses, the results are sufficiently robust to justify reclassification of the Rutaceae, with four subfamilies recognized in contrast to the traditional seven. This subfamily classification includes major rearrangements: (1) taxa within the subfamily Aurantioideae Horan. remain the same; (2) subfamily Cneoroideae Webb encompass the subfamilies Spathelioideae Engl. and Dictyolomatoideae Engl., Harrisonia R. Br. ex A. Juss. of the Simaroubaceae DC., Cneorum L. of the Cneoraceae Vest, and Ptaeroxylon Eckl. & Zeyh. of the Ptaeroxylaceae J.-F. Leroy; (3) subfamily Rutoideae Arn. include Ruta L. and Chloroxylon DC. of subfamily Flindersioideae Luerss.; and (4) subfamily Amyridoideae Link unite Flindersia R. Br. of subfamily Flindersioideae with subfamily Toddalioideae K. Koch and taxa previously included in subfamily Rutoideae.