Studies of reptile (nonavian reptiles) chromosomes began well over a century ago (1897) with the initial report on the description of sand lizard (Lacerta agilis) chromosomes. Since then, chromosome analysis in reptiles has contributed significantly to understanding chromosome evolution in vertebrates. Reptile karyotypes are also unique, as being the only vertebrate group where the majority of the species possess variable numbers of macro- and microchromosomes, which was first reported for iguanids and teiids in 1921. In addition, many reptiles have microchromosomes as sex chromosomes, highlighting their evolutionary significance, yet very little is known about their evolutionary origin and significance in shaping amniote genomes. Advances in genomic technologies in recent years have accelerated our capacity to understand how sequences are arranged within a genome. However, genomic and cytogenetic analyses have been combined for only 3 species to provide a deeper understanding of reptile chromosome evolution and sequence organization. In this review, we highlight how a combined approach of cytogenetic analysis and sequence analysis in reptiles can help us answer fundamental questions of chromosome evolution in reptiles, including evolution of microchromosomes and sex chromosomes.