The goal of this study was to detect antibiotic resistance and determine the ability of clinical isolates Klebsiella pneumoniae (K. pneumoniae) to form biofilms and detect the relationship between biofilm formation and antimicrobial resistance that depends on the specimen sources at the localized Baghdad Hospitals. This gram-negative rod bacterium is an opportunistic pathogen that can cause various illnesses in people and animals, such as important respiratory tract infections. For these reasons, our study included 87 isolates of K. pneumoniae from different clinical cases. The number and percentage of obtained isolates according to the sources distributed as specimens: 26(29.9%) urine, 25(28.7%) blood, 8(9.2%) stool, and 4(4.6%) sputum, as well as swabs:11(12.6%) burn, 9(10.3%) vagina and 4(4.6%) wound. The antibiotic susceptibility test results showed colistin, Imipenem, and meropenem were more effective against the isolates. The tissue culture plate and Congo red methods were used to evaluate biofilm formation. Finally, polymerase chain reaction was used to identify two genes linked with biofilm formation: MrkD and FimH. The isolates showed different abilities to produce biofilms based on clinical sources. The result appeared (97.7%) of isolates as biofilm producers from the following: 41(47.13%) strongly, 33 (37.93%) moderately, and 11 (12.64%) weakly. While only two isolates 2 (2.3 %) represented non-biofilm producers. 100 and 91% of the isolates, respectively, had the MrkD and FimH biofilm formation genes, according to molecular analysis. A recent study showed biofilm formation by K. pneumoniae strains isolated from blood specimens could form stronger biofilms. On the other hand, stool specimens formed weaker biofilm compared to them. According to this study, multidrug-resistant (MDR) K. pneumoniae strains' capacity to form biofilms and their antibiotic resistance profile is positively correlated. These could aid in developing therapeutic therapies for infections brought on by K. pneumoniae resistant to carbapenems, considered the "final line of defense" antibiotics. We can infer from this work that K. pneumoniae could be isolated from many sources and was MDR, as well as having the different capacity to build biofilm in various ways, especially in hospital cases of high antibiotic resistance. Keywords: K. pneumoniae, Biofilm formation, MDR, PDR, XDR, Congo red