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Previous experimental studies demonstrated that small drones (SD) had lower paternity share since they were not successful in mating with queens as large drones (LD) in the mating arena. However, it remains unclear whether spermatozoa of SD can compete in vivo with those of LD if SD have mating opportunity. We, therefore, tested the spermatozoal competitiveness of SD against LD by instrumentally inseminating the queens with varying proportions of semen from LD and SD. Sister queens from a Buckfast colony and LD and SD from 15 Caucasian colonies were reared synchronously as experimental test individuals. The virgin sister queens were randomly allocated into three semen composition groups. The queens in groups A, B and C were inseminated with an equal volume of semen (7.2 μl) collected successively from 6 LD and 6 SD (50%:50% treatment), 3 LD and 9 SD (25%:75% treatment), and 9 LD and 3 SD (75%:25% treatment), respectively. Once oviposition starts in mating nucs, the queens were introduced into field colonies to proceed to lay eggs. After 3 months, about 100 newly emerged worker daughters from each queen were individually collected from the colonies for paternity assignment. Five polymorphic microsatellite loci (A024, A079, A43, A113, and Ap226) were analysed in 144 drones that were used to inseminate 12 experimental queens and 908 offspring workers. The observed patriline frequencies of LD and SD were 67.0% and 33.0% in group A, 34.6% and 65.4% in group B, and 79.8% and 20.2% in group C, respectively. The patriline frequencies within each colony were noticeably skewed. LD that were reared in QRC sired more offspring, whereas SD that were reared in LWC had lower paternity shares. When all three semen composition groups were pooled, the overall observed patriline frequency of SD (40%) was found to be 10% less than the overall expected patriline frequency (50%). The results demonstrated that SD remained a little behind LD in sperm competition.