Sexual behavior in most moths is limited to a precise duration period of either the day or the night. These behaviors as well as the calling behavior are specific. Information on reproductive behavior is important in the chemical ecology studies. Experiments were conducted to determine the developmental biology, adult time emergence and calling behaviors of three populations of carob moth, Ectomyelois ceratoniae under laboratory conditions (29±1°C and 70±10% relative humidity in a 16-hr light-8-hr dark photoperiodism). Results revealed that mean egg developmental period in Saveh, Kerman and Arsanjan populations were 3.7, 3.19 and 3.6 days and mean total egg to adult developmental period of females were 39, 40.4 and 41.3 days and for males 37.4, 38.5 and 40.2 respectively. Adults began to emerge from the last hour of photophase with a peak occurrence at 1st hour of scotophase. The virgin females started the calling behavior on the first night after eclosion. Regardless of population, the majority of the calling on the side of the females occurred at third and fourth days after emergence with the peak occurring at the 8th hour of scotophase. The mean time spent in calling and the duration per calling bout by any of the three populations were equal, but there was no significant difference between the onset time of calling and number of calling bout by either Saveh or Kerman populations while being significantly different from Arsanjan population. Only a few females of the Saveh and Kerman populations exhibited calling during the first hours after the onset of photophase whereas many of Arsanjan females called at this time. This reflects that protandries occur in E. ceratoniae and for both sexes. Egg to adult developmental times were significantly different and females needed a minimum time for sexual maturation. Differences in calling behavior and biological characters under the same experimental and rearing conditions have a genetic basis which are the outcome of natural selection that are inheritable. These differences resulting from adaptation to different climatic conditions also suggest that the gene flow between these populations is limited.