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<i>Physalis pubescens</i> and <i>Physalis alkekengi</i>, members of the Physalis genus, are valued for their delicious and medicinal fruits as well as their different ripened fruit colors—golden for <i>P. pubescens</i> and scarlet for <i>P. alkekengi</i>. This study aimed to elucidate the pigment composition and genetic mechanisms during fruit maturation in these species. Fruit samples were collected at four development stages, analyzed using spectrophotometry and high-performance liquid chromatography (HPLC), and complemented with transcriptome sequencing to assess gene expression related to pigment biosynthesis. β-carotene was identified as the dominant pigment in <i>P. pubescens</i>, contrasting with <i>P. alkekengi</i>, which contained both lycopene and β-carotene. The carotenoid biosynthesis pathway was central to fruit pigmentation in both species. Key genes <i>pf02G043370</i> and <i>pf06G178980</i> in <i>P. pubescens</i>, and <i>TRINITY_DN20150_c1_g3</i>, <i>TRINITY_DN10183_c0_g1</i>, and <i>TRINITY_DN23805_c0_g3</i> in <i>P. alkekengi</i> were associated with carotenoid production. Notably, the MYB-related and bHLH transcription factors (TFs) regulated zeta-carotene isomerase and β-hydroxylase activities in <i>P. pubescens</i> with the MYB-related TF showing dual regulatory roles. In <i>P. alkekengi</i>, six TF families—bHLH, HSF, WRKY, M-type MADS, AP2, and NAC—were implicated in controlling carotenoid synthesis enzymes. Our findings highlight the intricate regulatory network governing pigmentation and provide insights into Physalis germplasm’s genetic improvement and conservation.