Ploidy levels and genome sizes have significant implications in plant evolution and crop improvement. Species of Lonicera L. have long been cultivated as medicinal, ornamental crops, or both. However, chromosome numbers, karyotypes, and DNA contents have only been documented in a few species, of which some controversies regarding basic chromosome numbers and karyotypes remain. This study analyzed the chromosome numbers and karyomorphology of 11 cultivars across four species and also the DNA content of 10 cultivars representing six species of Lonicera. Among them, the chromosome numbers of nine cultivars are reported for the first time. Results showed that the basic chromosome number of x = 9 was constant, and chromosome numbers of 2n = 18, 27, 36, or 54 were observed, suggesting that polyploidy exists in the genus. Five cultivars are diploid with 2n = 18; one cultivar is triploid, four are tetraploid, and one is hexaploid. The karyotypes of all studied cultivars are 3B or 3A, except Lonicera sempervirens ‘Crimson Cascade’ that is 2B based on the Stebbins’ asymmetry classification of karyotypes. The asymmetry index (A1) values vary from 0.47 to 0.60. The chromosome lengths range from 0.77 to 4.09 μm. Total karyotype lengths differ from 33.55 to 78.71 μm. The 1C-value of 10 cultivars varies 3-fold, ranging from 1.158 to 3.664 pg. Information gathered from this study could be valuable for improving breeding efficiency in the development of new cultivars of Lonicera with enhanced medicinal, ornamental value, or both.