Food safety knowledge (FSK) plays an important role in guiding food handlers (FHs) practices during food operations. The aim of this research was to investigate the level of FSK among FHs in hospitals' food services in Jordan. Up to 245 food handlers work in hospitals were purposefully selected from 6 public and private hospitals. A published FSK questionnaire included 120 items in 6 subscales was used to collect data. Most of the hospitals' FHs were male (91.84, n=225), of mean age 35.89 ± 9.26 years, with average experience of 12.55 ± 8.1 years, education of less than high school, and worked in public hospitals. The overall FSK was found to be "moderate" (71.20%). Food handlers were having the highest knowledge in the areas of "personal hygiene and hand washing" (80.87%), "health problems affecting FS" (86.10%), and "cross-contamination control and sanitation" (75.96%). FSK was "moderate" in "thawing, safe storage, transfer, catering, holding, heating, and re-heating" was (68.06%), "foodborne diseases and symptoms" (67.05%), and "poor" in "foodborne pathogens" (30.60%). Male FHs possessed higher FSK than female's counterparts (p=.036). There was a significant negative relationship between FSK and age (p=.044), positive relationship with previous exposure to FS training (p=.000), while there was no significant mean difference of FSK based on the years of experience or hospital sector (p=.090). One-way ANOVA with post-hoc revealed that FHs with higher educational qualification possessed higher FSK (p= .00). Food Safety Training is recommended for certain areas in food operations and foodborne pathogens.