Biodiversity is the source of energy and goals to maintain ecosystem for future. Present study of “Biodiversity and molecular characterization of Spider fauna of Khyber Pakhtunkhwa” was carried out in districts (Mardan, Swabi, Malakand, Swat, Dir Lower, Dir Upper, Peshawar, Charsadda, Kohat, Hangu, Karak, Lakki Marwat, Dera Ismail Khan, Mansehra, Battagram and Bajur Agency (F. A. T. A) from 2013 to 2016 in Khyber Pakhtunkhwa Province of Pakistan. All the species were collected during different times of seasons but mostly from March to November of above mention years. The specimens of spiders were collected through hand picking, beat sheet, pitfall trafs and sweep nets in different ecological niches. Preserved specimen were observed under stereomicroscope with supply of light, While pictures were taken by Leca MZ 16 microscope with power light and diagram ofmales and females genitalia were drawn with compound microscope with tube camera (Lecucida). Also, Molecular work was carried out, to extract DNA from four legs of spider species of subtribe Aelurillina (Salticidae) by DNA extraction kits. Four genes (28SrDNA, Wingless, 16S-ND1, COI) were amplified and sequenced by using primers. This study admits nine families of spiders (Araneae) contain twenty Six Genera and distributed in thirty seven species. The species are Aelureillus sp.nov, Aelureillus logunov, Bianor albobimaculatius, Bianor Pujabiicus, Carrohotus sannii, Castianeira sp.nov., Clubiona sp.nov., Crossopriza maculipes, Epocilla sp.nov., EvarchaArabeca, Hasarius adansonii, Heteropoida afghan, Hippasa paritita, Langona calcarata, Langona sp.nov., Lycosa sp.nov., Myrmarachni bicuirvata, Olios sanguinifrons, Oxyopies javana, Pardosa birmanica, Pardosa chaihraka, Pardosa flavisteirna, Peuceitia Arabica, Phelgra bresnieri, Plexippus Kondarensis, Plexippus paykulli, Plexippoides flavescens, Pseudicius admirandus, menemerus nigli (female first time described), steinaelurillus sp.nov., Thanatus fornicatius, Thyene Imperialis,Xysticus preuidocristatus, Yaginumaella sp.nov.,Zelotes spinuilosus. These Species are distributed in genera Bianor, Carrohotus, Castianeira, Chinattus, Clubiona, Crossopriza,Epocilla, Evarcha, Hasarius, Heteropoda, Hippasa, Olieos, Oxyopies,Lycosa,Myrmarachni, Pardosa, Peuceitia, Phlegra, Plexippous, Plexippoides,Pseudicius, Rhene, Thyene,Steinaelurillus, Xysticus, Yaginumaella, Zelotes and consitituted by the Families Corinnidae Clubionidae, Lycosidae Oxyopidae, Philodromidae, Pholcidae, Salticidae, Sparassidae, Thomisidae,. Present study determined 10 new to science species from genera Aelureillus, Castianeira, Chinattus, Clubiona, Epocilla, Evarcha, menemerus Female, Langona, Lycosa, Rhene, Steinaelurillus, Yaginumaella, some of them have been submitted for publication. The more diverse family is Salticidae with 14 genera and 23 species. The aim of the present study was to know the current status of unexplored region for spiders biodiversity with molecular phylogenetic studies to show the present relations of species with their ancestors. We recommend future work on Taxonomy, Biodiversity and Molecular Phylogenetic study of araneae and related arachnids of Pakistan.