Antimicrobial resistance was determined for 341 thermophilic Campylobacter jejuni isolates obtained from human clinical cases (n = 101), broiler products (n = 98), dairy cattle (n = 41) and wild birds (n = 101) with known multilocus sequence types (MLST) in Lithuania. The minimum inhibitory concentration (MIC) values for ciprofloxacin, tetracycline, gentamicin, ceftriaxone and erythromycin were determined with the agar dilution method. MIC values were compared with MLST types to find possible associations among isolation source, sequence type and resistance to antibiotics. The proportions of resistant strains were 94.2% (human), 95% (wild birds), 100% (broiler products) and 100% (dairy cattle) for one of the tested antibiotics. Most frequently, resistance to ciprofloxacin was observed (91.5%), followed by ceftriaxone with 60.4%, and tetracycline (37.8%). However only three C. jejuni strains were resistant to erythromycin (0.9%) and all tested thermophilic Campylobacter strains were sensitive to gentamicin. Most of the examined C. jejuni isolates (80.6%) showed resistance to at least one of three profiles: CIP+AXO (28.1%), TET+CIP+AXO (26.7%) and CIP (25.8%). Statistically significant differences in resistance to tetracycline were found between C. jejuni strains obtained from cattle (85.4%) and broiler products (64.3%) (P < 0.05). The majority (87.1%) of the tested strains from wild birds were resistant to ciprofloxacin (P < 0.05). The results showed that strains of novel ST's showed significantly lower resistance to ceftriaxone (P < 0.05). The ST-21 (CC21) (78.8%) was identified with significantly higher multidrug resistance relatively to other tested ST's in this study. Our results emphasize the high antimicrobial resistance of phylogenetically diverse C. jejuni strains isolated from different sources including specific genotypes of wild bird's strains in Lithuania. The results support the opinion that not only broiler products but cattle and wild birds may be a reservoir of resistant C. jejuni and stipulate a risk of spread or resistant bacteria. There is increasing need for broad surveillance and control measures to track changes and pathways of antimicrobial resistance of C. jejuni in epidemiologically distinct populations.