Background/Objectives: Sickle cell disease (SCD) is a multi-systemic hemoglobinopathy affecting many patients worldwide. Clinical manifestations include vaso-occlusive (VOC), aplastic, or hemolytic crises. Anemia of a complex etiology is also very common. However, detailed data regarding the level of anemia in Greek or Mediterranean populations with SCD/β-thalassemia are limited. Methods: We retrospectively investigated anemia in 53 SCD patients and described the molecular background regarding mutations in beta and alpha alleles in 49 SCD patients (49/53, representing 92.45% of the cohort). DNA analysis was performed in the Laboratory of Medical Genetics, National and Kapodistrian Athens University. Results: The median age was 50 years (range 18–79 years). A total of 20 patients (38%) were diagnosed with severe anemia (Hgb < 8 g/dL), and 6 patients had a very severe degree of anemia (Hgb < 7 g/dL). On the contrary, four patients had very mild or no anemia, depending on their sex. The median hemoglobin levels of the cohort were 8.72 g/dL (range 6.5–13.3 g/dL), while median reticulocyte levels were 183 K/μL (range 18.35–706 K/μL). We found that patients with the beta zero thalassemic allele had statistically decreased Hgb levels (mean Hgb 8.29 g/dL) and thus a more severe degree of anemia compared with SCD patients with a beta plus allele (mean Hgb 9.48 g/dL, p = 0.010 < 0.05, Student’s t test). Conclusions: Our genotype-stratified analysis characterized the molecular basis of anemia in a well-defined cohort of SCD patients. Clinical trials and future research are essential to investigate possible novel drugs for treating anemia and managing its long-term complications in SCD patients.