Introduction Acute lymphoblastic leukemia (ALL) has suboptimal survival rates for adolescents and young adults (AYA) as compared to children. Very limited studies have been conducted on AYA patients in India. This study aimed to identify the cytogenetic and immunophenotype characteristics of B-cell ALL (B-ALL) in AYA patients and determine its correlation with clinicopathological parameters in the Southern India region. Method The study was a prospective study conducted for three years, from June 2019 to May 2022, in India. Newly diagnosed 90 patients with AYA (13-40 years) ALL were recruited. A B-ALL diagnosis was made based on morphology with cytochemical stains and immunophenotype by flow cytometry (FCM). Cytogenetic analysis was also performed using karyotyping and fluorescent in situ hybridization to identify chromosomal aberrations. The cytogenetics results were correlated with immunophenotyping and clinicopathological characteristics. Variables were analyzed using the Mann-Whitney U test and Chi-square test using IBM SPSS Statistics for Windows, Version 20.0 (Released 2011; IBM Corp., Armonk, NY, USA). Results The mean age was 22.68 ± 8.06 years. It was observed that the most common structural chromosomal abnormality for AYA was t(9;22) in 14 (15%) cases, followed by 6q deletions in seven (8%) cases, t(1;19) in four (4%) cases, and t(12;17) and t(6;14) in two (2%) cases each. In addition, t(3;12), t(2;11), t(12;21), t(1;9), t(2;12), and t(X;10) were found in one (1%) case each. The most common numerical abnormality was hyperdiploidy (15; 17%), followed by hypodiploidy (10; 11%). Further, myeloid antigen expression of CD33 was the most common aberrantly expressed marker found in 20 (28%) cases, followed by CD15 in three cases (5%), CD13 in three (4%) cases, and CD11b in two (3%) cases. It was also observed that in Ph+ve cases, CD33 and CD13 were most commonly expressed in three (33%) and two (17%) cases, respectively. In contrast, in Ph-ve patients, their expressions were lesser at 17 (27%) and one (2%) cases, respectively. In addition, leukemia-associated immunophenotype pattern (LAIP) markers CD44 6 (86%) and CD123 5 (55%) were also found to be significantly associated with Ph+ve, whereas their values in the Ph-ve group were lesser at 25 (42%) and 9 (17%), respectively. Our data also showed that older age wassignificantly associated with Ph+ve with a median age of 30 years (p = 0.012). In comparison, the median age of Ph-ve was only 21 years. Conclusion Our study established that the incidence of cytogenetic abnormalities for AYA was consistent with previously reported data. This study reaffirms that Ph+ve cases have significant associations with MyAg (CD13), LAIP (CD123 and CD44), and older age for the South Indian population.