Urinary tract infection (UTI) is one of the common infections diagnosed in community and hospitals. Due to increasing antibiotic resistance amid uropathogens, it is significant to have community and hospital based knowledge of the microorganisms causing UTI and their antibiotic sensitivity pattern to select right course of therapy. The objective of present study is to isolate bacteria from patient samples suffering with UTI and specially to focus on isolation and characterization of UTI pathogens total of 25 urine samples collected from patients reported with UTI from University health Center, OU and RTC Hospital Tarnaka, Hyderabad. A total of 25 bacterial isolates were obtained by pure culture techniques based on morphological variation. All the 25 bacterial isolates were cultured on UTI specific medium (CLED agar).They were further characterized and identified by biochemical methods. It was found that 8 isolates were Eschericha coli, 7isolates were Klebsiella species, 3 were Proteus species, 3were Pseudomonas species and 4 were Staphylococcus spp. All the 25 bacterial isolates were characterized for antibiogram pattern using Gentamycin, streptomycin, norfloxcin, oflaxacin and amoxicillin. Percentages of isolates indicate E.coli as the dominant pathogen of UTI compare to others and the isolate which showed maximum resistant to antibiotic was subjected to molecular identification and it was identified to be E. coli.