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Lutein and its <i>cis</i>-isomers occur in a lot of plants, including a variety of flowers. In this study, lutein isomers were produced via iodine-catalyzed isomerization, and four <i>cis</i>-isomers (9<i>Z</i>-, 9′<i>Z</i>-, 13<i>Z</i>-, and 13<i>Z</i>′) were isolated by means of column chromatography and semipreparative HPLC. The structures of the 9′<i>Z</i>- and 13′<i>Z</i>-isomers were elucidated via NMR measurements. These compounds were used as standards for the HPLC-DAD-MS determination of the carotenoid composition of the flowers of 20 plant species, in which lutein and its geometrical isomers are the main components. The flowers showed great variation in their <i>cis</i>- and <i>trans</i>-lutein content, and also in the presence or absence of other carotenoids, such as violaxanthin, neoxanthin, β-cryptoxanthin, and β-carotene. Some of the investigated flowers were found to be rich sources of lutein without zeaxanthin.