Most recent studies on Lilium species have been descriptive observations based on FISH (fluorescence in situ hybridization) analyses or phylogenetic studies focusing on only one section without taking the evolutionary framework of Lilium into account. To better understand the taxonomic status, evolution, and karyotype diversity of the genus Lilium, we conducted FISH mapping using 35S rDNA sequences in 29 important wild Lilium species covering four sections of the genus in China. Their molecular phylogenetic relationships were analyzed based on the internal transcribed spacer (ITS) region. For 35S rDNA, the hybridization loci always existed in pairs on homologous chromosomes. The number of hybridization signals varied from 4 to 14 in diploid species, and the signals were mainly located on the proximal region of the short arm of chromosomes. Among all the sections, sect. Sinomartagon had the most 35S rDNA signals. Genotype grouping based on 35S signal loci was largely consistent with groups in the phylogenetic tree constructed using ITS sequences, and different species in a sect./subsect. shared some common molecular and cytogenetic features. The results indicated that sect. Martagon is monophyletic in a single clade; Lophophorum should be treated as a different taxon from Sinomartagon; Lilium taliense and Lilium wardii Stapf ex Stern show close molecular and cytological phylogenetic relationships; Lilium duchartrei Franch. is more distantly related to other sect. Sinomartagon species; Lilium lophophorum (Bureau & Franch.) Franch. is distinct from other Lophophorum species; Leucolirion species form two relatively independent groups (6a and 6b, with Lilium henryi Baker and Lilium rosthornii Diels in 6a); and Nomocharis-like Lilium species should be treated as a different taxon, like sect. Lophophorum. Based on previous studies and our findings, we discuss the evolutionary trends of Lilium with references to its geographical distribution. We propose that sect. Lophophorum is a relatively primitive group, and species in subsect. Leucolirion 6b may be more evolved than those in 6a. These findings provide a new reference for the taxonomy, phylogeny, and breeding of Lilium.