Landrace pumpkins occur in nature and their potential as source of pro-vitamin A may be investigated in order to be used in conventional plant breeding or biofortification programs, aiming to increase the total carotenoids and β-carotene contents. The objective of the study was to determine the total carotenoid, α-carotene, β-carotene and its isomers and contents in two landrace samples (A and B) of raw pumpkins (Cucurbita moschata) to verify its seed production potential. High Performance Liquid Chromatography and UV/Visible spectrophotometry were used to determine α-carotene, β-carotene and its isomers, and total carotenoid contents, respectively. All analyses were carried out in triplicate. The results showed mean total carotenoid contents of 404.98 in sample A, and 234.21μg/g in sample B. The α-carotene contents varied from 67.06 to 72.99μg/g in samples A and B, respectively. All E-β-carotene was the most abundant isomer found varying from 244.22 to 141.95μg/g in samples A and B, respectively. The 9 and 13-Z-β-carotene isomers were still found in low concentrations in both analyzed landrace samples. The content of β-carotene in raw sample A showed to be promising for the production of seeds for cultivation and consumption.