Staphylococcus haemolyticus is frequently associated with infections in hospitalized patients and acts as a reservoir for antibiotic resistance genes. The widespread use of vancomycin in treating serious infections has led to the emergence of heteroresistance to vancomycin in certain S. haemolyticus isolates. This study aimed to determine the rate of heteroresistant vancomycin-intermediate S. haemolyticus (hVISH) among 166 blood isolates collected from patients admitted to Jawaharlal Institute of Postgraduate Medical Education and Research between January 2017 and June 2018. E-test was done to determine vancomycin minimum inhibitory concentration (MIC). Heteroresistance screening was performed using brain heart infusion agar containing 4 mu g/mL vancomycin (BHIV4). Confirmation of heteroresistance was performed by population analysis-profile area under curve (PAP-AUC) analysis. Whole-genome sequencing was performed on eight isolates. All isolates were vancomycin-susceptible by E-test, while 18.7% exhibited heteroresistance by PAP-AUC. The sensitivity, specificity, positive predictive value, negative predictive value, and kappa agreement of BHIV4 with PAP-AUC were 64.5%, 97.8%, 87%, 92.3%, and 0.692, respectively. Mutational analysis of the eight isolates revealed variations in vraSR, tcaRAB, walKR, yycHIJ, rpoBC, lytSR, clpP, phoR, PBP4, msrR, cmk, and mprF operons, along with novel mutations in cell wall synthesis operons. The rate of hVISH isolation in our study is in agreement with other studies worldwide. As the sensitivity of BHIV4 is only moderate, it may be prudent to consider alternate antibiotics if MIC of vancomycin approaches the breakpoint. Furthermore, our analysis revealed multiple mutations within glycopeptide resistance operons, providing a better understanding of the molecular mechanisms underlying heteroresistance in S. haemolyticus and its implications for antimicrobial therapy and surveillance strategies.