Some mycoplasmas were isolated from most of the oropharyngeal swabs taken from 7-month-old layer chickens in Ipon but not from 1-month-old broiler chickens in Sitiawan in Malaysia. It was noted that 16 of the 18 strains isolated from one layer chicken flock were argineni catabolizing micoplasmas and identified as Mycoplasma gaolnarum through metabolism inhibition test. On the other hand, 26 of the 31 strains from another layer flock were a glucose fermenter, one of which was identified as M. synoviae. This is the first isolation of this species in Malaysia. An improvement was made on the ELISA technique in order to precisely detect antibodies against M. gallisepticum and M. synoviae. The improved technique employed antigens made from young cultures, blocked the antigen coated plates, and diluted test serum with skim milk containing buffer. A serological survey of chicken flocks in Malaysia using the improved ELISA technique together with a serum plate agglutination and hemagglutination-inhibition test that both M. gallispeticum and M. synoviae infections prevailed in layer chickens at high incidences but not in broiler flocks in the middle part of West Malaysia.