This study aimed to elucidate the clonal origin and evolutionary dynamics of T-cell prolymphocytic leukemia (T-PLL) using targeted next generation sequencing (NGS) of paired samples from diagnosis and relapse. DNA from both nonmalignant and tumor cells was extracted from sorted cell fractions obtained from 16 patients with T-PLL. NGS was performed using a customized Haloplex gene panel comprising 19 genes recurrently mutated in T-PLL (ATM and JAK/STAT pathway). Droplet digital polymerase chain reaction was performed to confirm mutations detected by NGS with low variant allele frequencies. Single-cell analysis of genomic DNA combined with cell surface protein markers was performed using the Mission Bio Tapestri Platform. The most frequently mutated gene was ATM (n = 10) followed by STAT5B (n = 7), JAK3 (n = 3), EZH2 (n = 3), BCOR (n = 1), and STAT6 (n = 1). Relapse samples were available for 9 of the 16 patients. Varying patterns of clonal shifts were observed between diagnosis and relapse (increase, decrease, both increase and decrease, and no change). The presence of pathogenic variants in ATM, EZH2, STAT5B, and JAK3 in both normal sorted B cells and clonal T cells was confirmed. Single-cell analysis revealed shared mutations in both nonmalignant B and clonal T cells in 1 case. A pathogenic variant within the ATM gene of potential germ line origin was observed in 1 case. T-PLL exhibits variable patterns of clonal evolution between diagnosis and relapse. Single-cell multiomics analysis reveals shared mutational signatures in both nonmalignant B cells and clonal T cells. The role of germ line ATM mutations in the pathogenesis of T-PLL should be further explored.