Transposable elements (TEs) are mobile genetic elements in the eukaryotic genome. They are divided into two classes (class I: retrotransposons and class II: DNA transposons) based on their structure and manner of transposition. TEs are major components of the eukaryotic genome and retrotransposons are especially abundant in higher-plant genomes. As retrotransposon insertions with high copy numbers are dispersed throughout the genome and are inherited genetically, insertion polymorphisms among crop cultivars have been used as molecular markers. Recently, we developed an efficient method for screening the long terminal repeats (LTRs) of retrotransposon families that exhibit high levels of insertion polymorphisms among crop cultivars using a next-generation sequencing (NGS) platform. This method focuses on the primer binding site (PBS) that is adjacent to the 5' LTR and has a conserved DNA sequence among different LTR retrotransposon families. Construction of a sequencing library through PCR amplification using the PBS conserved sequence allowed us to acquire a large number of LTR sequences and their insertion sites throughout the genome. From our data analysis, we screened the LTR sequences that showed high levels of insertion polymorphism among closely related cultivars. In addition, we identified the insertion sites of these identified LTR retrotransposon families at the genome-wide scale in a number of cultivars with an NGS platform, which enabled us to reveal the genetic relationships among the cultivars and acquire a number of molecular markers for cultivar screening. Our results indicated that the target sequencing of these retrotransposon insertion sites was highly effective for DNA genotyping and marker development without requiring any whole-genome sequence information. This review describes the genomic structure and evolutionary aspects of TEs and discusses the development of molecular markers based on retrotransposon insertion polymorphisms.