Genetic variability in modern crops is limited due to domestication and breeding. To investigate genetic variation in different populations, 216 tomato (Solanum lycopersicum L.) cultivars, hybrids, and elite breeding lines from four breeding programs were genotyped using single nucleotide polymorphism and simple sequence repeat markers. Of 47 markers analyzed, 72.3% were polymorphic in the whole collection of 216 genotypes and 51.06 59.57% showed polymorphisms in individual populations. However, genetic variation was narrow in all four populations, Nei's genetic distance varied from 0.0422 to 0.1135 between populations and from 0.0085 to 0.3187 between lines in individual populations. Cluster and principal coordinate analysis indicated that the four populations could be grouped into three clades. Lines from Shenyang Agricultural University and China Agricultural University population formed the first clade, lines from Beijing Vegetable Research Center were in the second clade, and lines from Nunhems were in the third clade. This was further supported by population structure analysis using STRUCTURE2.2, and suggested that a lack of germplasm exchange might exist among breeding programs. It rnight be the reason that the progress of developing new varieties with significant improvement of horticultural traits in China is slow in recent years.