Abstract: In sexually active male Japanese quail cloacal gland hypertrophy and foam production is androgen dependent and highly positively correlated with testis size and sexual activity. Nevertheless, the physiological functions of this gland in male reproduction are still a controversial issue. Therefore, the aim of this investigation was made to rule out the effect of cloacal gland size on semen production, sperm activities and fertility in both Heavy Body Weight (HBW) and White Feathered (WF) lines of Japanese quail. Sixty matured males and thirty females (8 weeks) from each lines of Japanese quail were randomly selected. Males were categorized into three groups (20/group) based on the increasing order of cloacal gland area. They were maintained in uniform husbandry condition with ad libitum feed and water at 14 h photo-schedule. Highest (11.04±0.20 and 9.6±0.41) semen production was recorded in birds having largest cloacal gland area and was positively correlated to the cloacal gland size in both the experimental quail lines. Metabolic activity by adopting Methylene Blue Reduction Time (MBRT) test and proteolytic activity by Acrosine Proteolytic Activity (APA) test of quail spermatozoa was found significantly different (p≤0.05) among groups and also revealed a positive correlation to the increasing area of the cloacal gland. In both the experimental lines, fertility was noted maximum (p≤0.05) in group III categorized by largest cloacal gland area. From this current study, it may be concluded that cloacal gland can be considered as external indicator or selection marker of testicular functions in male Japanese quail.