Short-chain fatty acids (SCFAs) are crucial products of microbial fermentation in the gut, known to play a significant role in maintaining gastrointestinal health and the overall well-being of animals. Betaine, a naturally occurring compound found in various plant and animal sources, has been widely used as a feed additive in poultry production due to its potential benefits, including improved growth, osmoregulation, and liver function. This study examines the possible effects of betaine supplementation on SCFA-producing bacteria in laying pullets raised in tropical climates, mainly focusing on SCFA-producing Lactobacillus and Bifidobacterium species. In total, 96 laying hens strain Lohmann aged three weeks were assigned to two dietary treatments. Dietary treatments included a basal diet (control) and a basal diet supplemented with 1.2 g.kg-1 betaine. The next-generation sequencing method of the 16S rRNA gene region V3–V4 was applied to view the bacteria abundance. Betaine supplementation showed significantly higher population levels of Lactobacillus_agilis, Lactobacillus_aviarius, Lactobacillus_ingluviei, Lactobacillus_johnsonii, and Lactobacillus_saerimneri compared to the control group (P<0.05). In Bifidobacterium_pullorum bacteria, betaine administration shows insignificant results compared to control. In conclusion, betaine supplementation appears to have a positive impact on promoting the growth of certain beneficial Lactobacillus species in laying pullets reared in tropical climates.