Fowl cholera is a contagious bacterial disease of poultry caused by Pasteurella multocida and has a significant economic importance worldwide. Fowl cholera prevention depends mainly on vaccination using live and inactivated vaccines, but they have many limitations. The present study aimed to prepare fowl cholera chicken embryo derived inactivated bacterin and to evaluate it by experimental infection and molecular evaluation. Fowl cholera chicken embryo derived inactivated bacterin prepared by in-vivo growing of Pasteurella multocida serotype 3 (P-1059) on chicken embryonating eggs, then inactivated by formalin (0.3%) and adjuvant added and then quality control parameters tested. The prepared bacterin was evaluated by experimental challenge with homologues and heterologous serotypes of Pasteurella multocida serotypes in comparison with commercial inactivated multivalent vaccine. Molecular evaluation of prepared bacterin was carried out using SDS-PAGE for in-vivo and in-vitro grown Pasteurella multocida serotypes. Results revealed that the prepared fowl cholera chicken embryo derived inactivated bacterin was free from bacterial and fungal contaminations and safe for use. Fowl cholera chicken embryo derived inactivated bacterin provided high protection rates with low mortalities against experimental infections with homologues and heterologous serotypes of Pasteurella multocida. Results of SDS-PAGE revealed that in-vivo grown Pasteurella multocida serotypes showed expression of additional specific bands (35kDa, 39kDa). It was suggested that 39kDa is a dominant structural protein in Pasteurella multocida grown in-vivo and play a role in antigenic cross protection among its serotypes. In conclusion, Fowl cholera chicken embryo derived inactivated bacterin is effective against infections of homologous and heterologous Pasteurella multocida serotypes.