Abstract: Hemoglobin concentration is a crucial hematological parameter that reflects the oxygen-carrying capacity of blood and serves as an indicator of general health and anemia prevalence. Variations in hemoglobin levels are influenced by several factors, including age, gender, nutritional status, and physiological conditions. Understanding these variations provides valuable insights into population health and helps in developing preventive strategies for anemia and related disorders. Materials and Methods: This retrospective cross-sectional study was conducted in El-Beyda City, Northeast Libya, using laboratory records collected between 2020 and 2015. A total of 1,200 subjects (600 males and 600 females) aged between 10 and 80 years were included. Hemoglobin levels were determined using an automated hematology analyzer (Sysmex KX-21). Participants were categorized into age groups: <20, 20–40, 41–60, and >60 years. Statistical analysis was performed using SPSS version 25, employing ANOVA and Pearson’s correlation to evaluate the relationship between hemoglobin levels, age, and gender. Results: The mean hemoglobin concentration was 14.1 ± 1.2 g/dL in males and 12.8 ± 1.1 g/dL in females, showing a statistically significant difference (p < 0.001). Hemoglobin levels tended to decrease progressively with age in both genders. The highest mean values were observed among individuals aged 20–40 years, while the lowest were among those older than 60 years. A weak but significant negative correlation was found between hemoglobin levels and age (r = –0.26, p < 0.01). The overall prevalence of anemia (defined as Hb < 13 g/dL in males and Hb < 12 g/dL in females) was 18.5%, higher in females (24%) than in males (13%). Conclusion: This study demonstrates a significant influence of both gender and age on hemoglobin concentration among the population of El-Beyda City. Females and older individuals exhibited lower hemoglobin levels, suggesting an increased risk of anemia in these groups. These findings highlight the importance of regular hematological screening and nutritional awareness programs, especially targeting women and the elderly, to prevent anemia and promote community health in Northeast Libya.