DNA molecular markers technique has been introduced as new approaches to study fungal phylogeny and evolution. Nowadays, fungal molecular systematics is a mature discipline, and the history of development and representative research approaches of fungal molecular systematics and the main attributions of these approaches to fungal systematics are discussed in this paper. The organisms studied by mycologists, fungi, are divided into three different groups. The slime moulds and oomycetes do not belong to the Kingdom of Fungi. The recent milestone is the AFTOL (Assembling the Fungal Tree of Life) project, in which a higher-level phylogenetic classification of the Fungi is proposed. The kingdom Fungi traditionally consisted of Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, but more recent classifications of the fungal kingdom now include Ascomycota, Basidiomycota, Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Glomeromycota, Microsporidia and several subphyla incertae sedis, including Mucoromycotina, Entomophthoromycotina, Kickxellomycotina and Zoopagomycotina. The concept of the different taxa in fungi has also been revised. Furthermore, the investigation of molecular markers technique influences the fields such as fungal species concept recognition, the connection of teleomorph and anamorph, and molecular fungal ecology, etc.