Contagious agalactiae caused by Mycoplasma agalctiae (M. agalctiae) implicated for important losses in small ruminant due to mortality, decreased milk production, and cost of treatment and prevention. There is a lack of information about the disease status in Palestine. In this study, a survey of M. agalctiae antibodies in sheep blood from Jericho city near the Jordan River in the eastern Palestine was conducted. A total of 611 randomly selected sheep of different ages were investigated from different farms in two regions in Jericho city during the period from December 2016 to March 2017 to. Blood samples were collected and serum was subjected to serologic examination for detection of antibodies against M. agalctiae major membrane lipoprotein of (p48) using enzyme-linked immunosorbent assay (ELISA, CHECKIT, IDEXX). The overall M. agalctiae seroprevalence rate was 11.5%. Regarding the distribution of the disease in the investigated regions in Jericho city; the seroprevalence rate of M. agalctiae in sheep was 13.5% in Al-Jiftlik and 4.4% in Fasa'il. This is the first report of M. agalactiae infection in Palestine. Further studies are required to determine other causes of contagious agalactiae and to include other regions in Palestine.