BACKGROUND: Calcium (Ca) and phosphorus (P) intake during brooding affects laying hens' later production and health, with availability varying among strains, necessitating further investigation of the factors influencing these differences. OBJECTIVES: This study aimed to compare the availability of Ca and P and related gene expression among 3 high-yielding layer strains during the first 6 wk and identify genes strongly associated with nutrient absorption. METHODS: Ninety pullets (1-d-old, female) from 3 strains [medium size, large egg layer (ML), light size, medium egg layer (LM), and dwarf, small egg layer (DS) weighted 31.533 ± 0.63 g, 39.367 ± 1.40 g, and 34.099 ± 0.64 g, respectively] were randomly assigned to 6 replicates of 15 birds each for 6-wk cage rearing. Feces were collected weekly to track the Ca and P availability. Initial and final body weights and tibial lengths were recorded to determine growth performance. Intestinal samples were collected to determine the gene expression of Ca and P transporters [transient receptor potential cation channel subfamily V member 6, calbindin D28k, Na+/Ca2+ exchanger 1 (NCX1), plasma membrane Ca-ATPase 1b, and sodium-dependent phosphate transporter IIb (NPt2b)] as well as tight junction proteins (claudin-2 and claudin-12). RESULTS: ML and LM pullets exhibited significantly greater body weight (443 g, 436 g compared with 319 g, P <
0.001) and tibial length (70.6 mm, 69.6 mm compared with 59.2 mm, P <
0.001) than DS. Notably, during the sixth week, the Ca and P retention in DS (0.847 g/wk compared with 1.648 g/wk, 0.662 g/wk compared with 1.141 g/wk) was significantly lower than that in ML, and in most weeks, DS exhibited the lowest Ca availability among the 3 strains. Gene expression analysis revealed higher expression levels of Ca transporters in the duodenum of ML and LM than in DS, whereas DS demonstrated elevated transporter expression in the jejunum. Furthermore, ML and LM exhibited more pronounced expression of tight junction proteins across most intestinal segments. CONCLUSIONS: The study indicated that expression of Ca and P transporter is highest in the duodenum, and duodenal NCX1, NPt2b were the genes most significantly positively correlated with the retention and excretion of Ca and P in pullets.