<p>The increasing global demand for natural rubber has prompted research efforts to focus on developing propagation techniques for the rapid production of <i>Hevea</i> clones on a large scale. This study aimed to examine the in vitro establishment of the <i>Hevea brasiliensis, H. guianensis, H. spruceana,</i> and RRIM 600 genotypes from zygotic embryos subjected to various treatments with plant growth regulators (PGRs). Furthermore, the morphoanatomical structure and expression profile of genes involved in latex biosynthesis (<i>REF1, REF3, REF7</i> and <i>SRPP1</i>), as well as in the formation of laticiferous vessels (<i>CDPK1, MPK3</i> and <i>WOX13</i>) were evaluated. The emergence and subsequent development of seedlings were observed to occur via different strategies. However, these treatments didn’t result in any observable morphological changes. The secretory structure exhibited distinctive cellular morphological characteristics between the species, as evidenced by the presence of articulated and anastomosed laticiferous vessels in <i>H. brasiliensis</i> and RRIM 600, articulated and non-anastomosed vessels in <i>H. guianensis</i>, and non-articulated, branched, laticifer cells with aggregated distribution in <i>H. spruceana.</i> Genes associated with latex biosynthesis and the formation of laticiferous vessels were expressed in seedlings belonging to all genotypes under study. The results demonstrate that the MS medium exhibited superior performance in the emergence of complete seedlings across all genotypes when compared to the MS medium with plant regulators. Furthermore, the combined morphoanatomical parameters and differential expression of the genes <i>REF3, SRPP1,</i> and <i>MPK3</i>, may serve as potential candidate genes for early selection of <i>Hevea</i> spp. species and the mass propagation of in vitro seedlings of “elite” genotypes, thereby facilitating the advancement of rubber tree breeding programmes.</p>