This study evaluates longitudinal and transversal intramuscular variations in muscle fiber and meat quality characteristics in bovine M. longissimus thoracis et lumborum (LTL). The LTL muscles (n = 5) from the left side of the beef carcass were cut at intervertebral segment intervals (between 1st thoracic vertebra (TV) and 6th lumbar vertebra (LV)). The pennation angle demonstrated an increasing trend from the anterior to posterior regions regardless of the medial (M-zone) and lateral (L-zone) regions (P < 0.05). The M -zone had a higher pennation angle than the L-zone in the TV and 1st LV (P < 0.05). The cross-sectional area (CSA) of muscle fibers, excluding type I, was larger in the posterior region than the anterior region (P < 0.05). A larger CSA of type I/IIA, IIA, IIAX, and IIX was observed on the lateral side than on the medial side of the 13th TV (P < 0.05). Fiber types were more oxidative (types I and IIA) in the anterior region and more glycolytic (types IIA/IIX and IIX) in the posterior region. Fat content was higher in the anterior region than in the posterior region (P < 0.05). The lowest redness, yellowness, and Warner-Bratzler shear force values were observed in the middle of the muscle, whereas the lightness value was lower in the posterior region regardless of the transversal region (P < 0.05). Therefore, bovine LTL muscles exhibit unique morphological properties and contribute to understanding meat quality associated with morphological and muscle fiber characteristics in relation to their intramuscular variations.