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Simple Summary: Current recommendations suggest setting two parallel cytogenetic cultures with 12-O-tetradecanoly-phorpol-13-acetate (TPA) and IL2+DSP30 as a mitogen to detect complex karyotypes (CKs) in chronic lymphocytic leukemia (CLL). However, studies comparing CK detection concordance between both methods in the same cohort are lacking. Herein, we evaluated the performance of two parallel cultures in a CLL cohort of 255 patients, specifically comparing CK detection (globally and in each individual patient). The CK detection rates and their prognostic impacts were similar for both mitogens. However, nearly one-third of CKs were only identified in one culture, mainly due to the detection of a normal karyotype or no metaphases in the other. In summary, the assessment of parallel cytogenetic cultures is the best strategy to detect CKs in CLL. Nonetheless, as IL2+DSP30 achieved the best performance, it should be prioritized above TPA if a single analysis is required to optimize cytogenetic assessment in routine practice. Current CLL guidelines recommend a two parallel cultures assessment using TPA and IL2+DSP30 mitogens for complex karyotype (CK) detection. Studies comparing both mitogens for CK identification in the same cohort are lacking. We analyzed the global performance, CK detection, and concordance in the complexity assessment of two cytogenetic cultures from 255 CLL patients. IL2+DSP30 identified more altered karyotypes than TPA (50 vs. 39%, p = 0.031). Moreover, in 71% of those abnormal by both, IL2+DSP30 identified more abnormalities and/or abnormal metaphases. CK detection was similar for TPA and IL2+DSP30 (10% vs. 11%). However, 11/33 CKs (33%) were discordant, mainly due to the detection of a normal karyotype or no metaphases in the other culture. Patients requiring treatment within 12 months after sampling (active CLL) displayed significantly more CKs than those showing a stable disease (55% vs. 12%, p < 0.001). Disease status did not impact cultures' concordance (κ index: 0.735 and 0.754 for stable and active). Although CK was associated with shorter time to first treatment (TTFT) using both methods, IL2+DSP30 displayed better accuracy than TPA for predicting TTFT (C-index: 0.605 vs. 0.580, respectively). In summary, the analysis of two parallel cultures is the best option to detect CKs in CLL. Nonetheless, IL2+DSP30 could be prioritized above TPA to optimize cytogenetic assessment in clinical practice. [ABSTRACT FROM AUTHOR]